Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP(+). An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.