The biocatalytic resolution of (R,S)-epichlorohydrin to (R)-epichlorohydrin by whole cells from a recombinant Escherichia coli BL21(ED3) expressing epoxide hydrolase activity in a series of organic solvent/buffer biphasic systems was studied. Isooctane was found to be the best organic solvent. The optimum volume ratio of the organic phase to the aqueous phase, reaction temperature, pH, substrate concentration, cell concentration, and reaction time were 7:3, 30 °C, 8.0, 574 mmol/L, 0.07 g/ml wet cells, and 45 min, respectively. Under the optimized conditions, a yield of 37.5% with 99.3% ee and an average productivity of 0.286 mol/(L·h) were obtained in a stirred reactor (1-L scale). Thus, the bioconversion was more efficient in the isooctane/buffer biphasic system than that in a monophasic aqueous system, where the substrate concentration and the average productivity of (R)-epichlorohydrin were improved by 55.2% and 98.6%, respectively.