Abstract: Determination of lactic acid and acetic acid of biological selenium vinegar and vinegar by using HPLC method were studied.C₁₈ as chromatographic analysis column, detection wavelength was 215 nm, the mobile phase was 2.5% NH4H2PO4 buffer, sample dilution was 30 times, the flow rate was 1.0 min/m L, the column temperature was 29 ℃, the best p H of the biological selenium vinegar was 2.5, while the best p H of the vinegar was 2.7, which could better separation and determination of lactic acid and acetic acid of biological selenium vinegar and vinegar, all testing completed within 4 min.When other analysis conditions were certain, p H was 2.5, the regression equation of acetic acid and lactic acid standard were y = 484.23x-81.373, R²= 0.9992, y = 441.37x-68.321, R²= 0.9979, respectively.When p H was 2.5, the regression equations of acetic acid and lactic acid standard were y = 484.61 x + 72.201, R²= 0.9993, y = 389.82 x + 30.432, R²= 0.9991, respectively. The relative standard deviation of this method was 0.00% ¹.07%, standard addition recoveries of biological selenium vinegar lactic acid and acetic acid standard were 100.00% ¹10.00%, 99.00% ¹00.83% respectively, standard addition recoveries of vinegar lactic acid and acetic acid were 100.00% ~ 101.00%, 100.00% ~ 110.00% respectively. The method has good repeatability, accuracy and precision, short detection time, therefore it could be applied in biological selenium vinegar and vinegar.