Abstract: Response surface methodology was used to optimize the fermentation conditions of chitinase by Pseudoalteromonas sp.DL-6. Firstly, the important factors influencing chitinase production were identified by the method of Plackett-Burman as colloidal chitin, fermentation temperature, and pH. The path of steepest ascent was applied to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were used to further investigate the optimal central levels and relationships of these factors. By using the quadratic regression model equation and analyzing the response surface of chitinase production, the optimal fermentation conditions were determined as followed:colloidal chitin 8.95g/L, peptone2g/L, shaking speed 130r/min, inoculums dose 2%, fermentation temperature 20℃, and pH 6.18, respectively.Under these conditons, the maximumtheoretic activity of chitinase was 57.95U/mL. After three verifications, the maximum theoretic value was consistent with mean value of verification test and the chitinase production was increased by 23.77% comparing to that before optimization.