Abstract: To efficient preparation of (R)-p-chlorostyrene oxide (pCSO), whole cells of E. coli/pveh1Z4X4-59 expressing a Phaseolus vulgaris epoxide hydrolase mutant PvEH1Z4X4-59 was used as biocatalyst to hydrolytic kinetic resolution racemic (rac-) pCSO. In the phosphate buffer (pH=7.0) system, the kinetic resolution of 250 mmol/L rac-pCSO catalyzed by E. coli/pveh1Z4X4-59 was perforemed, retaining (R)-pCSO with an over 99% enantiomeric excess (ees). Moreover, based on the effects of six nonionic surfactants on the EH activity and enantioselectivity of E. coli/pveh1Z4X4-59, Tween-20 was selected as the best reaction additive to construct a Tween-20/buffer system. In the optimized Tween-20/buffer (4%, volume fraction) system, the initial concentration of rac-pCSO catalyzed by 200 mg/mL E. coli/pveh1Z4X4-59 was significantly increased to 800 mmol/L (123.7 g/L), producing (R)-pCSO with 99% ees and 45.7% yield (theoretical yield was 50%). The space-time yield was reached 4.71 g/(L·h). Thereafter, 4.5 g (R)-pCSO was prepared with a total yield of 36.3% in extended 100 mL Tween-20/buffer reaction system.