A model compound 4-phenyl-2-butanone was used for studying the effect of glycerol in yeast Saccharomyces cerevisiae catalyzed reaction culture on the enantioselectivity for producing R- and S-4-phenyl-2-butanol under various reaction conditions. As the volume percentage of glycerol in the cell culture was 10%, the reaction gave a best R-enantiomeric excess (e.e.) value of 72.3% and a yield of 86.4% at the reaction conditions: a substrate concentration of 10μgmL−1, 25°C, an initial medium pH 10.0 but without controlling the pH, and without aeration. As the volume percentage of glycerol in the cell culture was 30%, the reaction showed a best S-e.e. value of 96.0% and a yield of 19.0% at the reaction conditions: a substrate concentration of 100μgmL−1, 34°C, pH 7.0, and without aeration. Substrate inhibition was shown with this reaction.