Production of (3S)-acetoin ((3S)-AC), an important platform chemical, is desirable but difficult to perform. An NADPH-dependent carbonyl reductase (Gox0644) from Gluconobacter oxydans DSM 2003 was confirmed to have a good ability to reduce diacetyl (DA) to produce (3S)-AC. In this work, the NADPH-dependent carbonyl reductase was expressed and purified. Glucose dehydrogenase from Bacillus subtilis 168 was coupled with the NADPH-dependent carbonyl reductase to produce (35)-AC from DA. Under the optimal conditions, 12.2 gl(-1)(3S)-AC was produced from 14.3 gl(-1) DA in 75 min. Because DA can be biotechnological produced, the two-enzymes coupling system might be a promising alternative for the (3S)-AC production. (C) 2013 Elsevier Ltd. All rights reserved.