Abstract: To screen and identify high chitosanase producing strains and to optimize the fermentation condition and analyze the enzymatic properties of the target strain. In this study, high chitosanase-producing strain was screened from the sediment of shrimp ponds in Beihai, Guangxi through primary and secondary screening. The strain underwent identification, the enzyme production process of the strain was optimized through single-factor, Plackett-Burman and orthogonal optimization methods. The enzymatic properties and products of chitosanase produced by the strain were studied. The results showed that the strain of Gxun-5.3.3 with high chitosanase production was obtained by screening and was identified as Bacillus cereus. The optimal components for the enzyme-producing medium of B. cereus Gxun-5.3.3 was maltose 5 g/L, yeast powder 3 g/L, chitosan 15 g/L, and the initial pH and fermentation time were 6.0 and 84 h, respectively. Under the optimized conditions, the chitosanase activity reached 62.03±1.18 U/mL, which was 55.58% higher before optimization (39.87±1.24 U/mL). The protease produced by strain Gxun-5.3.3 had maximum activity at 60 ℃ and optimum pH at 5.0, respectively. Metal ions Fe²⁺ and Mn²⁺ had a significant (P<0.05) activation effect on chitosanase, but Al³⁺ and Cu²⁺ had a strong inhibition. The enzymatic chitosan products were determined by TLC (Thin Layer Chromatography) as chito-oligosaccharides with polymerization degree 2~4, indicating that the chitosanase produced by the strain was acting on endo-oligosaccharides. This study provides a theoretical basis for the chitosanase production of B. cereus Gxun-5.3.3 and the enzymatic preparation of chito-oligosaccharides.