Objective Bacillus subtilis BS2, which can produce tetramethylpyrazine (TTMP) from glucose, was engineered by knockout of the 2,3-butanediol (2,3-BD) dehydrogenase gene (bdhA) and then regulated through the addition of 2,3-BD to enhance the TTMP yield. Results The bdhA of B. subtilis BS2 was disrupted to construct a TTMP-producing strain termed BSA. In microaerobic flask fermentation, the BSA strain produced 27.8 g TTMP/l. This was 6 g/l higher than that produced by the initial strain. Compared with that in BS2, the maximum yield of acetoin, which is a TTMP precursor, also increased from 11.3 to 16.4 g/l in BSA. The TTMP production by BS2 was enhanced by 2,3-BD supplemented to the fermentation medium. The maximum TTMP and acetoin yields were improved from 21.8 to 29.7 g/l and from 11.3 to 15.4 g/l, respectively, as the 2,3-BD concentration increased from 0 to 3 g/l. Conversely, the yields did not increase when the 2,3-BD concentration in the matrix was a parts per thousand yen4 g/l. Conclusions This study provides valuable information to enhance the TTMP productivity of mutagenic strains through gene manipulation and fermentation optimization.