Abstract: 【Objective】 In order to provide theoretical basis for revealing the biological function of flavonol synthase of Tetrastigma hemsleyanum Diels et Gilg in Huaiyushan, the flavonol synthase gene of T. hemsleyanum Diels et Gilg in Huaiyushan was cloned and analyzed by expression.【Method】 The core fragment of flavonol synthase gene was screened from the transcriptome database of plantlets of T. hemsleyanum Diels et Gilg in Huaiyushan. The flavonol synthase gene of T. hemsleyanum Diels et Gilg in Huaiyushan was cloned by reverse transcription PCR(RT-PCR) technique, sequenced by bioinformatics method and analyzed in organ expression by real-time fluorescence quantitative PCR.【Result】 The total length of flavonol synthase gene cDNA of T. hemsleyanum Diels et Gilg in Huaiyushan was 987 bp and the content of G+C was 47.92%. The flavonol synthase of T. hemsleyanum Diels et Gilg in Huaiyushan was made up of 329 amino acids with molecular weight of 37578.03 Da and isoelectric point of 5.39, which was hydrophilic protein. The secondary structure was composed of α-helix(27.66%), β-lamella(21.88%), irregular curl(50.46%). The flavonol synthase of T. hemsleyanum Diels et Gilg in Huaiyushan mainly existed in the endoplasmic reticulum. The evolution of flavonol synthase of T. hemsleyanum Diels et Gilg in Huaiyushan was closely related to Nekemias grossedentata, Vitis riparia and V. vinifera, especially to flavonol synthase of N. grossedentata, they had the highest phylogenetic relationship. The results of qRT-PCR showed that the expression of flavonol synthase gene was organ specific in the two cultivars of T. hemsleyanum Diels et Gilg in Huaiyushan. Huaiyu No.2 had the highest expression in the root and Huaiyu No.1 had the highest expression in the stem.【Conclusion】 The flavonol synthase of T. hemsleyanum Diels et Gilg in Huaiyushan has typical structural characteristics of flavonol synthase. Its amino acid sequence and nucleic acid sequence are highly similar to homologous species and highly conserved in evolution. There are tissue organ differences in the expression of flavonol synthase gene of T. hemsleyanum Diels et Gilg in Huaiyushan.