Optically active epoxides can be prepared by kinetic resolution of racemic mixtures using stereospecific epoxide hydrolases. To increase the bio-resolution efficiency of a sparingly water-soluble epoxide (glycidyl phenyl ether, GPE), we investigated the use of organic/aqueous two-phase system.Various conditions were systematically examined and optimized in shake flasks. Isooctanewas found to be the most suitable solvent as the organic phase. The phase volume ratio (φo/w) and biocatalyst concentration were shown to be sensitive parameters affecting both the reaction rate and the enzyme enantiospecificity in the biphase system. An isooctane/aqueous system was developed to overcome the low solubility and instability of GPE in the aqueous phase, resulting in a significant improvement of enatiomeric ratio (E-value) from 39.5 to 94.0 and an average productivity of 18.8mg GPE/(h g) biocatalyst to 48.9mg GPE/(h g) biocatalyst, respectively. Resolution of a 90.1g/l solution of racemic glycidyl phenyl ether in isooctane phase was successfully carried out in a mechanically stirred reactor (120ml), affording (S)-glycidyl phenyl ether in high (100%) enantiomeric excess with a yield of 44.5%.