Abstract: Water-fast silk fibroin films were created from regenerated silk fibroin by drying at a controlled temperature and humidity and degenerating with ethanol. To evaluate the biosafety of these silk fibroin membranes, cytotoxicity test in vitro, general allergic, delayed- type hypersensitivity assay, acute toxicity, pyrogenic reaction and hemolytic test on erythrocytes of rabbits were conducted. The results showed that: after in vitro cultured fibroblasts were treated with silk fibroin membrane leaching liquor, they proliferated appropriately at 92.3%, indicating the cytotoxicity of silk fibroin membrane leaching liquor was grade I; after repeated injections of 100% silk fibroin membrane leaching liquor, no mice presented generalized anaphylaxis; all the mice that received injections of 100% silk fibroin membrane leaching liquor survived, no acute general toxic reactions; the body weight of mice gradually increased within 7 days. After injection of 100% silk fibroin membrane leaching liquor into the ear vein of Japanese big eared rabbits, the body temperature of per rabbit increased by 0.2-0.5℃, but did not exceed the upper limit of 0.6, and 1.4℃ in total. The hemolysis test showed that the hemolytic rates of 100%, 50%, 25%, and 10% silk fibroin membrane leaching liquor were 4.02%, 2.57% , 1.56% , and 1.01% ，respectively, all rates were lower than 5% . In short, the data indicate that regenerated silk fibroin films exhibit good biosafety.