Abstract: At present, there is a lack of research on the effect of silk fibroin (SF) hydrogel degradation rate on the behavior of cells. To solve the problem, SF microspheres with or without protease were prepared through phase separation method, and were encapsulated into SF hydrogels photocrosslinked by ruthenium compounds. Through the proportion regulation of the microspheres, the effective control of SF hydrogel degradation rates could be achieved. Based on the construction of SF hydrogels with different degradation rates, NIH3T3 cells were selected to investigate the effect of SF hydrogel degradation rate on the proliferation of cells adhering on its surface. The hydrogel degradation rates were verified by the corresponding mass loss during the incubation period. Further researches were conducted on the effects of SF hydrogel degradation rates on the microstructure and compressive mechanical properties by using scanning electron microscopy and electronic universal testing machine. In addition, the effect of different hydrogel degradation rate on the proliferation of cells cultured on its surface was evaluated by CCK-8 method. Results showed that the degradation rate of SF hydrogel could be feasibly and effectively regulated. The degradation rate was proportional to the ratio of encapsulated microspheres with protease. After 6 days of incubation, the fastest degradation hydrogel lost about 40% of its original mass. The microscopic network structure of hydrogel was severely damaged, and the surface morphology has significantly changed from smooth to rough. The mechanical properties of the hydrogel gradually become hard and brittle from the initial soft elastomer. On the contrary, the weight loss of the slowest degradation hydrogel group was only about 10%, and the changes of microstructure and mechanical properties were relatively slight. Cell experiments show that the fastest proliferation of cells occurred on the SF hydrogel with moderate degradation rate at the encapsulation ratio of the microspheres without protease to the microspheres with protease of 7∶3. Related study provides a valuable reference for the controlling and constructing of tissue engineering scaffolds with appropriate degradation rates.