Bacillus amyloliquefaciens esterase (BAE) was applied to produce (R)-1-(3',4'-methylenedioxyphenyl) ethanol, a chiral drug intermediate. In this study, we improved the enantioselectivity of BAE by protein engineering instead of process engineering as used in our previous work. Saturation mutagenesis was carried out on eight positions of BAE based on structure modeling and substrate docking. A double substituted variant V10 (K358D/A396C) showed an excellent enantioselectivity without decreasing the activity. The functions of these two mutations (K358D and A396C) were investigated, revealing a synergic effect on the BAE enantioselectivity. Using the variant V10, enantiopure (R)-1-(3',4'-methylenedioxyphenyl) ethanol could be readily prepared in >97 % ee, affording a high space-time yield (123 g L(-1)day(-1)) and a high ratio of substrate/catalyst (40 g g(-1)) in 1-L reaction.