Reversed phase HPLC method was applied to determine organic acid in malic acid fermentation liquor in this paper.The chromatographic conditions are finally conformed to apply to all kinds of organic acid content determination in the fermentation.The conditions are as follows: COSMOSIL 5C18-PAQ column(4.6 mm I.D.× 250 mm) was used at 23℃.UV detection wavelength was 210 nm.Solution containing 1% acetonitrile and 20mmol/L KH2PO4 at pH 2.8 was used as mobile phase with flow rate of 0.5 mL/min.The seven organic acids in malic acid fermentation liquor were preferably separated under these conditions.The recovery ratio was 94.2%～102.7% and the method had good accuracy and precision.