In order to increase the yield of chitosanase, on the basis of single factor experiments, the fermentation medium of the mutant strain was optimized by response surface methodology. Seven components of the fermentation medium were analyzed by Plackett-Burman tests, and the three significant factors were determined as yeast extract powder, glucose and MgSO4·7H2O, and the reasonable range of factors was determined by the steepest climbing test, and then the medium composition was optimized by Box-Behnken response surface test. The results showed that the optimal fermentation medium was yeast extract powder 16.9 g/L, glucose 10.3 g/L, NaCl 5 g/L, K2HPO4 1.4 g/L, KH2PO4 0.6 g/L, MgSO4·7H2O 1.2 g/L and Tween-80 1.2 g/L. Under the optimal conditions, the chitosanase enzyme activity reached 10.57 U/ml, which was 11.77% higher than that before optimization.