The invention provides a preparation method of an ocular surface biofilm, which uses animal pericardium, intestinal membrane and adipose omentum as raw materials through degreasing, decellularization, multi-directional antigen removal, epoxy fixation, curved surface molding, surface Modification, cobalt-60γ-ray irradiation sterilization and other process steps. The ocular surface biofilm obtained by the method of the present invention has a curved surface similar to that of the eyeball, can be close to the eyeball, has no antigenicity, good histocompatibility, can participate in the healing process of ocular surface wounds, and is beneficial to wound healing; Good air permeability and bacterial filtration can play a good role in the protection and treatment of the ocular surface; it has good strength, can be pasted and sewed, and is easy to use. In addition, through the surface modification of drug sustained release, it can also be made into a therapeutic ocular surface biofilm that can release anti-infective drugs slowly.