Abstract: The metabolism Bacillus subtilis could produce acetoin and 2,3-butanediol,and a new method was established in this paper to detect the method of acetoin and 2,3-butanediol in fermentation broth by high performance liquid chromatography. The results of determination of acetoin and 2,3-butanediol by different chromatographic columns and flow matching ratio were compared and the detection conditions were optimized. The content of acetoin and 2,3-butanediol in the fermentation broth of Bacillus subtilis was further determined.The results showed that the Bio-Rad Aminex HPX-87H column was used to complete the sample detection,and the mobile phase was 0.005 mol/L of sulfuric acid solution,flow rate was 0.5 mL/min,column temperature was 28℃,refractive index detector was used,and the sample test was completed within 30 min.The correlation coefficients of the standard curve of acetoin and 2,3-butanediol were 0.9997 and 0.9998,respectively,the precision standard deviation was 1.76% and 1.74%,the stability standard deviation was 1.2% and 1.6%,the recovery rate was 98.93%~102.10%,and the relative standard deviation was ≤ 2%. This method was simple and accurate,the detection target concentration was 50 g/L,the test results were good on reproduction. The contents of acetoin and 2,3-butanediol in fermentation solution was negatively correlated,and the content of acetoin was decreased with the increasing of 2,3-butanediol,which was suitable for the detection of acetoin and 2,3-butanediol in the fermentation broth of Bacillus subtilis.