Acetoin is one of the bio-based platform chemicals and its optically pure isomers are important potential intermediates and precursors in the synthesis of novel optically active materials. ( 3R )-acetoin could be synthesized via enzymatic catalysis, whole-cell catalysis and fermentation. In this study a marine strain of Bacillus subtilis was isolated to produce optically pure ( 3R )-acetoin with glucose as carbon source. The effects of nutrients on the formation of ( 3R )-acetoin and conversion of glucose to (3 R )-acetoin were evaluated by Plackett–Burman design, and the fermentation medium was optimized by central composite design. The impact of oxygen supply on the production of (3 R )-acetoin was studied at different aeration rates. Under the optimal conditions, 83.7 g/L ( 3R )-acetoin with an optical purity of 99.4% was achieved by fed-batch fermentation, and the conversion of glucose to (3 R )-acetoin was 91.5% of the theoretical value. The results indicate the industrial potential of this strain for ( 3R )-acetoin production via fermentation.