A preparation method of separation membrane is provided. First, a polyimide composition including a dissolvable polyimide, a crosslinking agent and a solvent is provided. The dissolvable polyimide is represented by formula 1: wherein B is a tetravalent organic group derived from a tetracarboxylic dianhydride containing aromatic group, A is a divalent organic group derived from a diamine containing aromatic group, A′ is a divalent organic group derived from a diamine containing aromatic group and carboxylic acid group, and 0.1≤X≤0.9. The crosslinking agent is an aziridine crosslinking agent, an isocyanate crosslinking agent, an epoxy crosslinking agent, a diamine crosslinking agent, or a triamine crosslinking agent. A crosslinking process is performed on the polyimide composition. The polyimide composition which has been subjected to the crosslinking process is coated on a substrate to form a polyimide membrane. A wet phase inversion process is performed on the polyimide membrane.