Abstract: Culture and separation was made by using α-cyano-3-phenoxybenzyl acetate as the only carbon source after yeast enrichment. Further screening was performed by chiral HPLC analysis. Two yeast strains (S11 and S25) producing esterase with high enantioselectivity and conversion were isolated. Esterases were stable and remained activity at pH 3.0. Enantiomeric excess of (S)-α-cyano-3-phenoxybenzyl alcohol both reached 95%, the conversion of substrate reached 50% and 46% , respectively. The process of growth and partial enzymatic properties of S11 was investigated. Experiments demonstrated that it was a membrane-associated enzyme found in the cell debris.