The present invention relates to a multi-layered anti-adhesion barrier, particularly to a multi-layered anti-adhesion barrier comprising a nanofibrous structured base layer electrospun from a hydrophobic, biodegradable, biocompatible polymer and a polymer layer formed by coating a hydrophilic, biooriginated polymer on the base layer, and a method for the preparing the same. The multi-layered anti-adhesion barrier of the present invention can solve the problems of the conventional gel, solution, sponge, film or nonwoven type anti-adhesion systems, including adhesion to tissues or organs, flexibility, physical strength, ease of handling (ease of folding and bending), etc., offers improved user convenience. With a nanofibrous structure, the multi-layered anti-adhesion barrier of the present invention effectively blocks the infiltration or migration of blood and cells and promotes the healing of wounds. It is not torn or broken when folded or rolled and can be easily handled using small surgical instruments. Thus, it can minimize a foreign body reaction when used in various surgical operations.