The techniques for improving the stereoselectivity of asymmetric reduction of 3-oxo ester to chiral 3-hydroxy ester catalyzed by yeast cells were studied. Ethyl 4-chloro-3-oxobutanoate was chosen as the model substrate, and the effects of heating and enzyme inhibitor pretreatment of yeast cells on the stereoselectivity of the reduction reaction were investigated in detail. The results showed that the heating pretreatment and preincubation with allyl alcohol could remarkably improve the stereoselectivity of the S-(D)-product, respectively. The stereoselectivity of the S-form product increased with increasing temperature and treatment time in the heating pretreatment. Using allyl alcohol as enzyme inhibitor, the stereoselectivity of the S-form product also increased with increasing the concentration of allyl alcohol. Under appropriate pretreatment conditions, the ee of (S)-ethyl 4-chloro-3-hydroxybutanoate in the asymmetric reduction could reach as high as 97%.