One Night Kiss is an action-adventure game based on the anime series Blood+ created by Production I.G, set after episode 7 of the original series. The game is set in the fictional town of Shiki-Newtown, which has recently been terrorized by attacks from monstrous bat-like creatures called Chiropterans.

The player controls both the anime's main protagonist Saya Otonashi, a high-school girl who is capable of killing the Chiropterans, and Aoyama, a detective with the city police investigating the strange attacks. Saya wields a katana, while Aoyama uses guns as his weapon of choice. The player switches between the two characters automatically as the story proceeds.

Each character's chapters are divided into adventure parts and action parts. In adventure parts, the player walks around exploring, speaking with other characters, and looking for information. In action parts, the player must fight off the Chiropterans or other foes in closed-off arenas.