This game features eight playable characters, with four starting preset teams of two and unlockable individual character modes. When playing with the character pairs, one character fires when unfocused, while the other switches in and fires when focused, with a reduction in movement speed. The two shot types can be drastically different. The individual characters, with the exception of Remilia and Youmu, do not change shot types while focusing/unfocusing.

Imperishable Night features Last Spells. For the enemy, a Last Spell is a bonus Spell Card which the player can challenge without the option to use bombs and without the risk of losing lives. On the player side, Last Spells are secondary "bomb" Spell Cards, which last longer and do much more damage than normal bombs at the expense of two bomb stocks. Player Last Spells can only be used immediately after being hit, with a grace period of less than 1 second.

Additionally, there is a "Time Orb" system which affects the story/score, as well as a gauge which tracks how human/youkai (normal/focused) you are and determines how you can collect more time orbs.

A human and youkai team set off along a forest path to investigate. Along the way, they encounter a firefly (Wriggle Nightbug) and a night sparrow (Mystia Lorelei), who are quickly defeated. Next, they meet a half-beast (Keine) who is protecting a Human Village from youkai. She mistakes the team for enemies and attacks them. After the team defeats her, Keine indicates that the person who switched the moons is in the bamboo forest. In the bamboo forest, they encounter a character (either Reimu or Marisa) who demands to know why the team has been freezing time and causing an imperishable night.

Finally the team reaches the mansion of the perpetrator. But once inside, they find that the mansion is guarded by a pair of rabbits (Tewi and Reisen). The rabbits are defeated, but not before disorienting the team. Confused, the team must choose which path to take (one leads to the real moon and the other leads to the fake moon). After picking the right path, they are able to meet the perpetrator of this mischievous scheme, Kaguya. Kaguya is a moon princess in hiding who has taken the real moon in order to sever the link between the earth and the moon. The team restores the real moon and Kaguya challenges them to face the "5 impossible requests."