Gru, the former supervillain, has now settled into family life as the adoptive father of three girls, Margo, Edith, and Agnes. He has left behind a life of crime and is attempting to launch a legitimate business with Dr. Nefario and the Minions. However, Gru's peaceful life takes an unexpected turn when he is abducted by Lucy Wilde, an eccentric but capable agent of the Anti-Villain League (AVL). The AVL seeks Gru's help to apply his unique villainous insights to their cause, as a new and powerful criminal has stolen a secret Arctic laboratory containing a mutating chemical compound called PX-41.

Reluctantly teaming up with Lucy, Gru goes undercover to investigate The Paradise Shopping Mall where the AVL suspects the culprit may be hiding. As Gru begins to adjust to his role as a spy, he and Lucy embark on a series of comedic investigations, donning outrageous disguises, and surveilling the various shopkeepers who could potentially be the mastermind behind the PX-41 theft. While the professional side of his life becomes increasingly hectic, Gru also faces personal challenges, the most pressing being his daughters' desire for a mother figure and the budding romantic feelings Gru starts to develop for his quirky partner Lucy.