The acetoin microorganism fermentation system was prepared by biological method. The method to determine acetoin by high performance liquid chromatography was studied. The fermentation broth was pre-treated by centrifuging and filtering with 0.22 μm microporous membrane. A column of Hypersil NH2（300 mm×4.6 mm，5 μm）with an mobile phase containing acetonitrile and water(90∶10,V/V) was used for the analysis of acetoin in fermentation system, the flow rate was 1 mL/min, the column temperature and detector temperature was 35 ℃,the chromatography was completed in just 25 min. The recovery rate was 95.96%～101.2% and the relative standard deviations 1.69%～2.09%. The method was proved to be a rapid and effective quantitative analysis method with simple pretreatment and high sensitive to determine the acetoin in fermentation broth, which was fit for guiding real-time monitoring the fermentation process.