Acetoin is a natural flavor and mainly used as a food additive and important intermediate to synthesize novel optically active compounds. In this work a new acetoin producer, Bacillus subtilis DL01 was isolated from samples of sea sediment, and its growth properties and acetoin productivity were explored. The experiments showed that the new strain can grow well in a simple fermentation medium only containing sugarcane molasses, (NH4)(2)HPO4 and MnSO4. When the initial concentration of sugar was 200 g/l in molasses, 61.15 g/l acetoin (58.2 g/l D-acetoin) was obtained with a productivity of 0.807 g/(1 h) by a batch fermentation under stage-enhanced agitation and constant aeration of 0.4 vvm. By contrast, 76 g/l acetoin (60.9 g/l D-acetoin) could be obtained with a productivity of 1.00 g/(1 h) and a yield of 0.421 g/g if 210 g/l glucose was used. The strain DL01 demonstrated high sugar tolerance, high acetoin production, and low oxygen requirement. (C) 2015 Elsevier Ltd. All rights reserved.