Abstract: In order to improve the enzyme production ability of strain Bacillus cereus B03 quickly and efficiently,the fermentation medium of Bacillus cereus B03 producing β-lactamase was optimized by response surface methodology. Firstly,the single factor experiments were conducted to study the different enzyme sources and concentrations,different nitrogen sources and concentrations,different metal ions and concentrations,sodium chloride,dipotassium hydrogen phosphate,temperature,pH,inoculum and liquid loading. Influence,then design the Plackett-Burman experiment to screen out three significant factors affecting enzyme production:Temperature,pH,inoculum. On this basis,design the Box-Behnken center combination test and response surface analysis method to determine the optimal enzyme fermentation conditions. The results showed that the optimum fermentation medium was 20 g/L glucose,20 g/L yeast extract,NaCl 2 g/L,MgSO₄·7H₂O 0.2 g/L,K₂HPO₄·3H₂O 4 g/L. The optimum enzyme fermentation conditions were fermentation temperature 37 ℃,pH7.3,inoculum 3%,and liquid volume 50 mL/250 mL.Under this optimized condition,the enzyme activity of the strain producing β-lactamase was 113278.7 U/mL,which was 1.28 times that of the enzyme activity before optimization(88792.7 U/mL). This study would provide a reference for further industrial development using strains with stable traits and high production of β-lactamase.