Abstract: A HPLC method was developed for determinating the contents of Kaempferol-3-O-β-D-glucopyranoside, Kaempferol-3-O-β-D-galactopyranoside, and Quercetin-3-O-β-D-glucopyranoside in the leaves of Yunnan Diospyros kaki in different harvest periods.The extraction conditions were 45% ethyl alcohol, the ratio of liquid-solid of 20∶ 1, extraction time of 90 min for 4 times.The HPLC elution was carried out using methanol-0.4% phosphate system at a flow rate of 1.0 m L/min, the injection volume was 10 μL, and the detector wavelength was set at 265 nm.The RSD values of the three flavonoids were all less than 1% and the average recoveries were all above 99%.This method showed good separation effect and high stability, and could be used for determining the contents of the three flavonoids in the leaves of Yunnan Diospyros kaki in different harvest periods.The result showed that the accumulation of them in leaves of Yunnan Diospyros kaki exhibited regularity, and the contents were increasing along with fruit growing.