Abstract: A high performance liquid chromatography (HPLC) method was established for the simultaneous determination of L-ascorbic acid and D-isoascorbic acid. Under the chromatographic conditions, 0.05 mol/L phosphoric acid solution∶methanol (98∶2) was used as the mobile phase, the ultraviolet detection wavelength was 245 nm, the column temperature was 25 ℃, and the flow rate was 0.70 mL/min. The standard curves of L-ascorbic acid and D-isoascorbic acid displayed a good linear relationship (r=1.0). Precision (RSD) was 0.6%. The recoveries of L-ascorbic acid and D-isoascorbic acid were 90.83%~92.52% and 91.93%~92.99%, respectively. The method has the advantages of simple operation, fast extraction, high sensitivity and good recovery. The content of L-ascorbic acid was 8.24 mg/100 g, and no D-isoascorbic acid was detected. The method can be used to determine the contents of L-ascorbic acid and D-isoascorbic acid in other fruits and vegetables similar to Nanking cherry.