In order to improve the production of protease from Bacillus cereus, the fermentation medium composition and culture conditions were optimized. By single factor experiment, the effects were studied, including the optimum additions of carbon and nitrogen sources, the adding amounts, fermentation temperature, inoculation age, shaking speed, medium volume, medium initial pH, fermentation time, and inoculation amount on protease production. On this basis, Plackett-Burman was to evaluate the effects of nine variables, and selected significant factors, namely peptone addition amount, fermentation temperature and medium volume. The optimal fermentation condition was obtained by Box-Behnken and response surface analysis. The optimal conditions of peptone addition amount, fermentation temperature and medium volume were 7.9 g/L, 26.7 ℃ and 56.4 mL, respectively. Under the optimal conditions, the protease activity was up to 753.67 U/mL, 2.02 times higher than the initial protease activity (249.50 U/mL).