The invention discloses a method using an ice crystal template method to prepare a three-dimensional biological scaffold with a regular lamellar structure and application of the method. The method includes: sequentially subjecting fibroin fibers obtained after degumming silkworm cocoons to dissolving, filtering, dialysis and centrifuging, concentrating to obtain a silk fibroin aqueous solution, using the ice crystal template method to prepare an ice crystal/fibroin scaffold, using a constant cooling speed to perform freezing treatment, and using freeze drying to remove the regular lamellar ice crystals in the ice crystal/fibroin scaffold so as to obtain a fibroin scaffold, serving as the three-dimensional biological scaffold with the regular lamellar structure, with the regular lamellar structure. By the arrangement, the method has the advantages that the scaffold prepared by the method is excellent in biocompatibility and mechanical performance and evidently increases the proliferation performance of human cells and the directional differentiation performance of stem cells, and accordingly the bone tissue repairing effect of the biological scaffold is increased; the method is promising in application prospect in fields such as tissue engineering, drug sustained release, hemostatic materials and filtering membranes.