Abstract: M FBKL3.0018 selected from medium temperature Daqu was taken as the research object. The activity of esterifying enzyme in the fermentation liquor was viewed as examining index. Single factor experiment,Plackett-Burman experiment and response surface experiment were applied to opptimize the fermentation culture condition of esterifying enzyme produced by FBKL3.0018. The effect of carbon source,nitrogen source,inorganic salt,initial pH,fermentation temperature,rotate speed,culture volume,inoculation volume and fermentation time on the production of enzyme were investigated. It was turned out that the optimal medium was:2%beef extract,6% sucrose,0.2% CaCl₂,0.15% MgSO₄·7 H₂O,initial pH4.5,culture temperature 31 ℃,rotate speed 160 r/min,culture volume 45 mL/250 mL,inoculation volume 9%,and incubation time 96 h. Furthermore,culture temperature,time and sucrose,which had obvious effects on esterifying enzyme production,were selected to conduct response surface optimization experiments. The optimized conditions were fermentation temperature 31 ℃,fermentation time 96 h,sucrose concent ration 6%,under the condition of optimization,the activity of esterifying enzyme was 355.62 U/mL,2.67 times higher than that before optimization(133.12 U/mL). At the same time,compared with the predicted value(368.82 U/mL),the fitting rate was up to 96.4%,which indicated that the established regression model was reliable.