Abstract: Objective: The technique of producing cholesterol oxidase by the strain of Bacillus cereus SG03 in a 10 L fermenter was optimized, in order to provide the basis for further application of the strain.Methods: The effect of pH of the culture medium, stirring rate and DO on the production of cholesterol oxidase in the broth by Bacillus cereus SG03 was studied in a 10 L fermentor.Results: The optimum fermentation conditions were initial pH at 6.8, stirring rate at 250 r /min and DO at 80%.Based on the results, a strategy of two stage DO manipulation was proposed, controlling DO at 90% before the 20 h and at 20% from the 20 h to at the end during the fermentation.The maximum COD activity in the broth and production efficiency reached 1.38 U /mL and 0.03 U / ( mL·h) , respectively improved30.2% and 20.0% higher than with one stage manipulation.