A method for determination of organic acids and glucose in fermentation broth of Valsa mali var.mali by HPLC was established.The Atlantis C18 column(150 mm × 4.6 mm,3.5 μm) was used.Mobile phase was 5 g / L NH4H2PO4(pH2.5).The flow rate was 0.6 mL/min,and the temperature of column was 30℃.Organic acid and glucose in samples were determined by ultraviolet visible detector(UV) and refractive index detector(RID),respectively.They were separated effectively at the chromatographic condition.The average recovery was in the range from 96.2% to 102.2%,and the relative standard deviation(RSD,n = 5) was in the range from 0.26% to 0.75%.By this method,organic acids and glucose can be easily,rapidly and effectively determined within 15 minutes.