The invention provides a preparation method of a duramater/spinal dural transplanting substitute which is obtained by repeated freezing and thawing of dural tissue, rolling and cracking of cells, crosslinking fixed protection, accellular antigen extraction, dense surface fibrosis modification, packaging and sterilization, and has the advantages of simple method, wide raw material sources, cheap raw materials, and low cost. The prepared dural substitute completely removes components of cells and other antigen components simultaneously when protecting dural tissue natural structure and properties, is good in biocompatibility, free of immune rejection, safe and reliable, good in mechanical performance, and easy in clinical operation, can meet the needs of defect repair, has the function of promoting tissue regeneration as a loose surface is beneficial to the tissue fluid adsorption, active factor enrichment, and growth of blood vessels and cells, and has the advantages of rapidness in fusion with a host, biodegradable absorption, and good repair effect. The animal test shows that the defect can be completely repaired without brain or spinal fluid leakage, or adhesion with brain tissue, and significant rejection is not found.