Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (S)-acetoin and facilitate this building block for a series of chiral pharmaceuticals derivatives, we have developed a systematic approach using in situ-NADH regeneration systems and promising diacetyl reductase. Under optimal conditions, we have obtained 52.9 g L−1 of (S)-acetoin with an enantiomeric purity of 99.5% and a productivity of 6.2 g (L h)−1. The results reported in this study demonstrated that the production of (S)-acetoin could be effectively improved through the engineering of cofactor regeneration with promising diacetyl reductase. The systematic approach developed in this study could also be applied to synthesize other optically active α-hydroxy ketones, which may provide valuable benefits for the study of drug development.