The invention discloses a ply tissue engineering corneal frame and a manufacturing method and an application thereof, relating to a ply tissue engineering corneal frame of the biomedicine. Compared with the existing materials, the invention provides a ply tissue engineering corneal frame and a manufacturing method and an application thereof, which has abundant resources, high transparency, good biocompatibility, thorough decellularization and strong safety, and the performance approaches to that of a fresh cornea, so that the ply tissue engineering corneal frame can be accepted by majority of patients and applied clinically for a long term. The ply tissue engineering corneal frame is an animal derived decellularized ply corneal stroma sheet and does not contain cellular constituents; collagenous fibers are tidily arranged, and gaps are regular; corneal light transmittance is 80-95%, and tensile strength is 2-5N/mm<2>. The ply tissue engineering corneal frame can be served as the substitute for various donor materials for corneal transplantation and can be used for treating a series of diseases of corneal trauma and chemical burn, corneal tumor and a series of hyperplastic diseases, a series of diseases of corneal neovascularisation and scar, corneal immune diseases, a series of diseases caused by corneal transplantation exclusive reaction and other keratopathy.