In order to investigate the difference of active substances of Ampelopsis grossedentata leaves at different harvest periods and development stages, the three different development stages including bud tip, tender leaf and old leaf from May to September were employed to determine the polyphenol, soluble sugar, total amino acids and five flavonoids. The principal component analysis and cluster analysis were carried out to characterize the differences of active substances. The results showed that the contents of active substances were significantly different among different harvest periods and development stages. Concretely, the contents of dihydromyricetin(DMY), taxifolin, polyphenol and DMY isomer were higher in May, and the contents of bud tip and tender leaf were higher than those of the old leaf. The contents of myricitrin and soluble sugar were higher in May, July and the contents of old leaf and tender leaf were higher in August, September than those of the bud tip. The content of myricetin was higher in August and September, and the content of bud tip was higher than those of the old leaf and tender leaf. The content of total amino acids was higher in May, and the content of old leaf was higher than those of the bud tip and tender leaf. Moreover, three principal components were extracted by principal component analysis with the total cumulative variance of 93.456%. The comprehensive scores of bud tip, tender leaf and old leaf decreased in order, and the scores of different harvest periods at the same development stage were May, June, September, July, and August in descending order. The 15 samples of Ampelopsis grossedentata were divided into three groups by cluster analysis, and each group had specific quality characteristics, which were corresponding to bud tip, tender leaf, and old leaf respectively. The comprehensive scores of bud tip and tender leaf groups were significantly higher than those of old leaf. In summary, this study recommended bud tip and tender leaf of Ampelopsis grossedentata from May and June as excellent raw materials.