Following the events of the previous film, Gru (Steve Carell) has taken on the role of an agent for the Anti-Villain League (AVL) alongside his wife Lucy (Kristen Wiig). Their latest mission involves stopping the schemes of Balthazar Bratt (Trey Parker), a former child star who portrayed a villain in the now-canceled show "Evil Bratt." After experiencing a decline in fame during his adolescence, Bratt has embraced his old persona to become a true super-villain. Gru successfully prevents Bratt from stealing a pricey diamond, but fails to apprehend him, leading to the AVL's new and harsh director Valerie Da Vinci (Jenny Slate) firing both Gru and Lucy after numerous unsuccessful attempts to capture Bratt.
Reluctantly, they inform their children—Margo (Miranda Cosgrove), Edith (Dana Gaier), and Agnes (Nev Scharrel)—of their dismissal, assuring them that new opportunities will arise. Meanwhile, feeling disheartened, Dr. Nefario has encased himself in carbonite, and many of Gru's Minions leave when he chooses not to return to a life of villainy. Lucy finds it challenging to adjust to her new responsibilities as the kids’ mother.
The next morning, an unexpected visitor named Fritz (Steve Coogan), a butler for Gru's long-lost twin brother Dru, arrives. He reveals that Dru, who resides in a far-off land called Freedonia, is eager to meet Gru. In shock, Gru consults his mother (Julie Andrews), who explains that after divorcing Gru's father, they had agreed to raise one child each. Eager to meet his brother, Gru's family travels to Freedonia, where they discover Dru's surprising wealth, attributed to their successful pig farming business.
Simultaneously, the Minions fall into trouble after breaking into a talent show site and are subsequently arrested. Bratt, undeterred, manages to steal the diamond once again, plotting to use it to power a massive robot he built to exact revenge on Hollywood for canceling his TV series. As Lucy spends time with the children in Freedonia, she learns that Dru, feeling inadequate due to their father's past as a notorious super-villain, hopes Gru will show him the ropes of villainy. Gru, however, is reluctant to return to his villainous roots.
Despite their differences, the brothers bond over a ride in their father’s high-tech car. Back at home, Lucy tries to balance her responsibilities and faces a situation with a young boy named Nico who has a crush on Margo during a cheese festival, while Edith and Agnes hear tales of a mystical unicorn rumored to be in a nearby forest. Although Gru attempts to convince Agnes that unicorns are mere fantasies, he ultimately refrains out of love for her excitement.
On a quest to find the unicorn, Agnes and Edith unexpectedly encounter a one-horned goat that Agnes affectionately names “Lucky.” Meanwhile, the Minions, growing increasingly hungry, follow a pizza delivery boy into a studio, resulting in their arrest, yet they take control of the prison but long for Gru, leading them to escape.
Realizing that Bratt has regained possession of the diamond, Dru persuades Gru to partake in one final heist to infiltrate Bratt’s hideout, with Gru secretly aiming to recover the diamond for AVL reinstatement. After several obstacles, they successfully reclaim the diamond and are saved by Lucy, who had secretly followed them. Learning about Gru’s true intentions, Dru feels betrayed, prompting Gru to leave in anger.
At the same time, Bratt, disguising himself as Lucy, kidnaps the children and again acquires the diamond, which drives Gru and Dru to set aside their quarrels and work together. With his robotic device fully operational, Bratt wreaks havoc in Hollywood as planned. Lucy heroically rescues the children while Gru and Dru work together to battle the robot using their father’s car’s weaponry. Gru is knocked unconscious during the conflict, but Dru, embracing his inner villain, manages to destroy the robot’s core. When Gru regains consciousness, he confronts Bratt in an exaggerated dance-off, ultimately trapping him in his own gum and pushing him out of sight.
Reunited with the Minions, Gru and Dru now can celebrate their victory. In the aftermath, Gru and Lucy are reinstated at the AVL, and the family comes together to enjoy their time at home. Lucy is finally acknowledged as the children’s mother, while Dru, yearning for a life of villainy, steals Gru's ship, prompting Gru and Lucy to give them a five-minute head start before chasing after them.
