Abstract: In order to achieve the high-efficiency fermentation of cholesterol esterase, Pichia pastoris was selected as the research object, and its fermentation conditions were optimized by single factor and orthogonal experiments. The results showed that the optimum medium was yeast extract 1.0%, peptone 1.5%, glycerin 1.0%, KH₂PO₄ 1.18%, K₂HPO₄ 0.3%, biotin 4×10^-5%, YNB 1.34%. Under the condition of initial pH7.0, fermentation temperature 30 ℃, fermentation time 96 h, the enzyme activity could reach 11.21 U/mL. And it was 3.65 times higher than before. The results of enzymatic properties showed that the optimum pH and reaction temperature of the enzyme were 8.0 and 50 ℃, respectively. Zn²⁺ had a strong inhibition on the enzyme. The cholesterol esterase had stable enzymatic properties and could provide technical basis for large-scale production in the future.