In this study, whole cells of Saccharomyces uvarum SW-58 were applied in an aqueous-organic solvent biphasic system for the asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate to ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate [(R)-2]. The results of reduction in different aqueous-organic solvent biphasic systems showed that dibutylphthalate provided the best compromise between the biocompatibility and the partition of substrate and product among the solvents tested. To optimize the reaction, several factors such as reaction pH, temperature, shaking speed, volume ratio of the aqueous phase to the organic phase and ratio of biomass/substrate were investigated. It was found that the change of these factors obviously influenced the conversion and initial reaction rate, and had a minor effect on the enatiomeric excess of the product. Under the optimal conditions, 85.0% of conversion and 85.2% of enatiomeric excess were achieved. The bioconversion in the biphasic system was more efficient compared with that in the monophasic aqueous system, and product concentration as high as 54.6 g/L was reached in the organic phase without addition of co-enzyme.