The story of Twilight Syndrome: Saikai focuses on four Japanese teenagers: Yuri, her sister Masa, and their friends Atsushi and Aya. It all begins when the youngsters hear rumors about spirits contained in the school building. At night, Yuri and her friends go into the school and play a spirit-summoning game. Suddenly, real ghosts appear and scare the teenagers away. The next day, Yuri is almost ready to believe it was all a dream... but naturally it wasn't. The dark secrets of the lost souls are going to be revealed only to those which possess a special sense - the Twilight Syndrome..

The third entry in the Twilight Syndrome series is also a horror adventure, sharing most gameplay elements with its predecessors. The gameplay here is very minimalistic: the player can navigate the 3D models of the heroes (several characters can be controlled at once, as a group) through the limited areas (also rendered in 3D), searching for clues and escaping from danger. There is no other interaction, no usable items, and no combat.

Dialogue and action choices will pop out during many situations. Some of these choices do not influence the narrative, while others might change the following dialogue, and also lead the young heroes to premature death.

An additional gameplay mode (beside the story mode) is free-roaming exploration for Yuri, who can explore the school and its surroundings at night and gather more stories from the ghosts she encounters.