"Assassin Classroom" centers on Class 3-E of Kunugigaoka Junior High School, a group of students tasked with assassinating their alien teacher, Koro-sensei, before graduation. Koro-sensei, a tentacled, superhumanly fast being, has already destroyed 70% of the moon and threatens to annihilate Earth unless the students can kill him within a year. The Japanese government offers a 10 billion yen reward to whoever succeeds, incentivizing the students to take on the challenge.

The students of Class 3-E are considered the school's outcasts, relegated to a dilapidated building on a mountain due to their poor academic performance. Koro-sensei, despite being their target, dedicates himself to improving their grades, life skills, and self-confidence. He employs unconventional teaching methods, blending assassination training with traditional academics, and fosters a supportive environment where the students grow both academically and personally.

The series explores the dynamics within Class 3-E as the students develop unique assassination strategies, often reflecting their individual personalities and strengths. Key characters include Nagisa Shiota, a quiet and observant student who excels in stealth and strategy; Karma Akabane, a rebellious and highly intelligent student with a knack for combat; and Kaede Kayano, a cheerful and athletic girl with a mysterious past. Each student undergoes significant growth, learning to confront their insecurities and work together as a team.

As the story progresses, the students face external threats, including rival assassins and government agents, all vying to eliminate Koro-sensei. The narrative delves into Koro-sensei's origins, revealing his tragic backstory and the reasons behind his destructive capabilities. The students must grapple with moral dilemmas, questioning whether they can truly kill a teacher who has become a mentor and friend.

The series balances humor, action, and emotional depth, with Koro-sensei's eccentric personality providing levity amidst the high-stakes assassination plot. Themes of redemption, perseverance, and the value of education are woven throughout, as the students strive to overcome societal prejudices and prove their worth. The story culminates in a climactic confrontation, testing the bonds formed between Koro-sensei and his students while resolving the central conflict of his impending destruction of Earth.