A carbonyl reductase from Gluconobacter oxydans (GoCR) exhibited good activity and strict enantioselectivity for the asymmetric reduction of a series of halogenated acetophenones, resulting in the corresponding S-alcohols. For efficient synthesis of alcohol products, an economical and satisfactory substrate-coupled cofactor regeneration system was constructed employing isopropanol as the co-substrate to regenerate NADH in situ. In the presence of 2.0molareq. of isopropanol, 500mM o-chloroacetophenone was reduced to (S)-1-(2-chlorophenyl)-ethanol with >99% ee and a high conversion rate of 96% by recombinant Escherichia coli BL21 cells overexpressing GoCR. In this reaction system, a slight excess of isopropanol as hydride source could drive the reduction reaction to near completion due to the speculated forming of an intramolecular hydrogen bond between the Cl group and the new obtained alcohol moiety of (S)-1-(2-chlorophenyl)-ethanol. Furthermore, other tested halogenated acetophenones at 100 or 200mM substrate load were also reduced at 71–96% conversion, affording S-configuration alcohols with >99% ee. Homology modeling and molecular dynamics simulation were performed to uncover the structural basis for the excellent enantioselectivity of GoCR toward halogenated acetophenones. These results imply the high potential of GoCR in the production of halogenated 1-phenylethanols, such as (S)-1-(2-chlorophenyl)-ethanol, an important chiral building block with wide application in pharmaceutical chemistry and fine chemicals.