A method of reverse-phase high performance liquid chromatography(HPLC) for simultaneous and rapid determination of oxlate, malic, lactic, citric and succinic acid was established. The results showed that the recoveries of nonvolatile organic acid reached above 85.37% by phosphorous acid extracting under a water bath at 80 ℃ for 30 min. Using Welch Ultimate, AQC18 column with KH2PO4 (0.02 mol/L, pH3.0) and methyl alcohol as mobile phase at the detection wavelength of 215 nm, the nonvolatile organic acid in tobacco were well separated and their contents were well determined with relative standard deviations being 0.58% 1.99% and detection limits being 0.030.80 g/mL.