Four members of the Din Republic's spy team Lamplight embark on a mission to capture an enemy spy. Although they struggle to work together due to their clashing personalities, Thea, Monika, Erna, and Annett complete their objective. While enjoying a short break, the four girls encounter a woman named Matilda, who claims to be Annett's mother. As they get to know Matilda, the team is dragged into an escalating conflict that tests their teamwork and allegiances.

Meanwhile, the remaining Lamplight members notice that the dispatched squad has yet to report back. Worried about their allies, Klaus and Lily set out on an emergency expedition to locate their missing teammates. As the two uncover troubling information about rising tensions in the area where their friends last were, they must act quickly to reassemble the team before the mission reaches the point of no return.

Spy Kyoushitsu 2nd Season was released on Blu-ray and DVD in two volumes from October 25, 2023, to December 22, 2023.