The simple and exact method for the determination of the six substances existing in the fermentation was founded by employing high performance liquid chromatography.The chromatographic separation was performed on Aminex HPX-87H with mobile phase of 0.05 mmol·L^-1 of Sulfuric acid at a flow rate of 0.5 ml·min-1.Refractive index detector was chosen,and the column temperature was 15℃.In this seperation condition,a good linear relationship exits between the peak area of six substances and their concentrations with correlation coefficient ranging from 0.9991～1,and the recovery is form 99.34% to 101.11%.By the analyzing the samples,concentration of accessory production such as acetate,butyrate and the reactant glucose were entirely in a low level,meanwhile,the yields of the butanol,acetone and ethanol are close to the normal level.Suitable separation among the six substances were achieved,and the whole operation was consumed within 45 minutes.