The series focuses on Misaki Asano, a young woman assigned to the Future Foundation's 6th branch, dedicated to murder investigations in the midst of "The Tragedy", as she is teamed up with fellow investigator Takumi Hijirihara, a secret serial killer who only kills other killers, for the honor of Mukuro Ikusaba.

"[4] Misaki Asano (麻野 美咲, Asano Misaki) – The main character, a new recruit to the 6th Division of the Future Foundation who "striv[es] to bring light in a world ridden with despair", and is assigned to the Special Case Bureau as an investigator, specializing in murder investigations despite her dislike of gore.

Unbeknownst to Takumi, the Future Foundation leadership is well aware of his identity, doing nothing about him due to deeming his activities as beneficial to their cause.

Super High School Level Soldier) of Hope's Peak Academy and the overarching antagonist of the series, the original Ultimate Despair (with her sister Junko Enoshima), who massacred Takumi's and Shujii's middle school on her own when they were children, unknowingly respectively driving them to become serial killers of other serial killers as young adults.

In the third volume, Mukuro is apparently revealed to be alive (after her apparent death in the 2010 video game Danganronpa: Trigger Happy Havoc) and a prisoner of the Future Foundation, until it is revealed to not be Mukuro but a cult of people who have had plastic surgery to appear identical to Mukuro raiding the facility under command by Shuji so that Takumi would be forced to repeatedly kill visages of his idol.