tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl reductase exhibits excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) to synthesize (3R, 5S)-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3R, 5S)-CDHH was constructed in organic solvents. A solution of 10% (v/v) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, > 99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1M (S)-CHOH, compared with 98.7% yield and > 99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, > 99% ee and 4.08 mmol L-1 h(-1) g(-1) (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3R, 5S)-CDHH in mono and biphasic media.