Abstract: Natural spider silk has superior mechanical properties, and its various forms have excellent antibacterial activity, biocompatibility, and good thermal conductivity. It is of great significance to develop natural biological drug-loaded materials with good biocompatibility for reducing the strong immunogenicity reaction of patients. By exploring the dissolution and membrane-forming methods of spider silk in different solvents, the reaction conditions were optimized, hexafluoroisopropanol was used as the dissolution solvent, the material ratio was 1∶1 (mg∶mL), the spider silk was dissolved at 60℃ for 8 h, and the membrane was formed by solvent casting, and the drug release in vitro of rhodamine B (RhB) as the model drug was explored with the correlation characterization. The cytotoxicity of the material was tested by extraction method. The spider fibroin membrane extract co-culture with cells showed that the material had no potential cytotoxicity to mouse embryonic osteoblasts cells. The drug-loaded spider silk fibroin membrane prepared by this method has high yield, uniform thickness and simple operation, which provides a simple and feasible technology for the dissolution of natural spider silk protein and the preparation of membrane agent.