Abstract: The present invention provides a mutant strain of Bacillus subtilis SFA-H31 and includes an optimized method for producing of acetoin using this strain. The advantage of this method to produce acetoin has been defined by its high-yield without mixture with diacetyl or 2,3 butanedinol, both of which are usually accompanied with acetoin in other strain. This Bacillus subtilis mutant strain has been deposited in China General Microbilogical Culture Collection (CGMCC) and the accession number is CGMCC No. 1869, in which the process of fermentation for acetoin production is composed of (1) strain activation, (2) seed culture, (3) fermentation, (4) quantification of substrates and products. Based on current method, the concentration of acetoin in the ferment broth can reach to 35-55 g/L and the conversion rate of glucose to acetoin is in the range of 40-50%.