Abstract: Objective:In order to increase the protease activity by optimization of the fermentation medium of Stenotrophomonas maltophilia (Kx-7) in this study.Methods:firstly, the categories and concentration scales of carbon source, nitrogen sources and surfactant affecting the protease activity of Kx-7 were confirmed by single factor experiments.Based on this result, Box-Behnken design was used to optimize significant factors affecting protease activity.Finally, the second-order equation model for protease activity was established and fermentation medium suitable to protease production by Kx-7 was obtained.Results:Fructose, Casein and Triton X-100 were three major factors, the optimal concentrations were 19.15g/L, 4.05g/L and 5.1mL/L, respectively.Protease activity of Kx-7 could be increased by more than 1.2 fold (from 146.43U/mL to 324.56U/mL) after optimization.Conclusion:The protease activity of Kx-7 could be increased largely by the optimized fermentation medium through response surface methodology, which could be expected for mass production.