Sometime after the events of the second game in the Professor Layton series, Professor Layton and the Diabolical Box, the renowned archaeologist and puzzle master Professor Hershel Layton and his young apprentice Luke Triton find themselves reminiscing about one of their earliest adventures, set three years prior to the events of the previous game. The story begins when Layton receives an invitation from his former student, Janice Quatlane, to watch her perform in an opera at the historic Crown Petone opera house, located on the White Cliffs of Dover. The opera's plot revolves around the legendary lost Kingdom of Ambrosia and its mysterious secret to eternal life, which is believed to be revealed when the kingdom's queen finally returns. Janice suspects that recent strange occurrences, such as the disappearance of girls from London and the odd behavior surrounding the composer's new work, are somehow linked to the opera’s storyline.
The opera itself, conducted by the composer Oswald Whistler, features a complex contraption called the Detragon, an elaborate one-man orchestra machine that immerses the audience in its musical performances. As the show concludes, a mysterious figure appears among the crowd, announcing that the audience must now participate in a deadly game, with the prize being the secret of eternal life and the punishment for failure being death. Panic ensues as some audience members attempt to escape; however, the floor beneath them suddenly opens up, causing them to vanish without a trace. Inspector Grosky tries to apprehend the mysterious puppet master, but the man cleverly inflates and floats away, escaping while taking Grosky with him.
It is soon revealed that the Crown Petone is a ship, which detaches from the cliffs and sails away. The mysterious figure reveals a series of timed puzzles designed to eliminate the participants one by one, leaving only the cleverest to claim the prize. Thanks to Layton’s exceptional puzzle-solving skills, he, Luke, Janice, and nine others quickly deduce the answers, navigating the deadly challenges. Eventually, the remaining contestants abandon the ship via lifeboats, as the vessel explodes in the background, signaling the end of that perilous chapter.
The following morning, the group arrives on an island that appears to be the legendary realm of Ambrosia, identified by the presence of a carved stone seal nearby. As they explore, they must evade a pack of wolves and make their way toward the island’s central castle. During their journey, Layton, Luke, and Janice are separated from the others but ingeniously craft a makeshift helicopter, which allows them to reach the castle swiftly. Inside, they solve the final, crucial puzzle of the contest, which guides them to the last room, but Layton chooses to investigate more of the castle while Janice, Luke, and two other contestants — Marco Brock and child prodigy Amelia Ruth — proceed into the final chamber.
Meanwhile, on the mainland in London, Emmy Altava, Layton’s trusted assistant, learns about the events unfolding on the island. She quickly departs for the remnants of the ship, accompanied by Inspector Grosky, to follow the trail. In the castle, Layton discovers remnants of Melina Whistler’s belongings, along with a mysterious piece of sheet music titled A Song of the Sea. There, he encounters Melina herself, who appears to be having an internal argument with herself — a sign of her own fractured mind. At the same time, Luke, Janice, Brock, and Amelia find themselves trapped behind bars, confronting the revelation that the mysterious man orchestrating everything is none other than the formidable Jean Descole, a recurring nemesis of Layton and Luke. Descole orders Amelia to be taken to him, trying to manipulate the situation to his advantage. Fortunately, Emmy and Grosky arrive just in time to rescue the group and Layton, who then reunite with Melina.
It turns out that Melina is actually Nina, one of the girls who had gone missing from London. Amelia, who is declared the winner of the deadly contest, is soon strapped into a machine linked to the Detragon, and she confesses that there is no true Elixir of Life. Layton explains the brutal truth: the Detragon isn’t a device to grant eternal life but a sophisticated machine capable of copying a person’s personality and downloading their memories into another’s brain. Whistler, in collaboration with Descole, had kidnapped girls from London to use the Detragon to implant their loved ones’ memories into new bodies, effectively keeping them alive forever. Nina’s fate was to have her memories transferred into Janice’s body without her knowledge. Melina, the true queen of Ambrosia, had sought Layton’s help after discovering her father’s sinister plan and the truth about her own identity.
As Descole’s dark plans begin to unravel, he captures Melina and reveals his true ambition: to use the Detragon, along with her singing voice, to awaken the island of Ambrosia by playing two melodies—the Song of the Stars and the Song of the Sea. However, Descole’s attempt to activate the melodies fails, inciting him into a furious rage. In his desperation, Descole commandeers the Detragon to transform into a giant excavator robot called the Detragiganto, which rampages across the island, trying to uncover the legendary city of Ambrosia by force. As chaos unfolds, Layton, Luke, and Melina make their escape from the collapsing castle, flying away in their makeshift helicopter.
In the midst of the destruction, Melina attempts to stop Descole, but he pushes her off the Detragiganto, leaving her hanging perilously from the robot. Luke bravely rescues her, while Layton engages in a dramatic duel with Descole on top of the rampaging machine. During the fight, Melina sings again, this time revealing the hidden Song of the Sun, which Layton recognizes as the third melody needed to revive Ambrosia. With her voice, the ruins of the lost kingdom finally rise from the depths. Descole, overwhelmed by anger, lunges at Layton in a misguided attempt to claim the city for himself, but his assault damages the control panel and causes the giant robot to malfunction and self-destruct. Descole falls off and disappears into the chaos, leaving Layton, Luke, and Melina to escape the wreckage.
After the island’s salvation, Melina chooses to leave her false identity behind, relinquishing her connection to Janice’s body, and bids farewell to her father, Layton, and Luke. Her spirit departs, while Janice, now fully herself again, sings in honor of Melina’s memory—her voice carrying the message of hope and eternal life through stories told across generations. As dawn breaks, Layton reflects on the true meaning of eternal life, which, he suggests, is found in the legacy of stories and memories preserved by people who remember the lost kingdom of Ambrosia. Luke wonders if Melina might have been a reincarnation of Ambrosia’s queen, to which Layton responds with a legend: "Ambrosia will rise again when its beloved queen returns." The adventure concludes with a message of hope that life, memory, and legend endure beyond the bounds of age and death.
