In this study, a full length cDNA of flavanone 3hydroxylase (F3H) was cloned from the leaf of Ampelopsis grossedentata using RACE technique, and named AgF3H (accession number JX087441). The full length cDNA was 1 323 bp, containing a 1 092 bp open reading frame (ORF) encoding a protein consisting of 363 amino acids， with 2 typical functional domains and 5 conserved motifs of dioxygenase superfamily. Sequence comparison and phylogenetic tree analysis showed that the amino acid sequence of AgF3H had considerable similarity with those of other species, and had the closest evolutionary relationship with the F3H protein of Vitis vinifera, Vitis amurensis and Vitis rotifera. Real time PCR showed that the expression level of AgF3H gene decreased gradually during the maturity of leaves. The highest expression level emerged in buds while the lowest expression level emerged in young stems. Correlation analysis revealed positive correlation between AgF3H gene expression and the content of dihydromyricetin, the main flavonoid component in A.grossedentata. After the overexpression vector of AgF3H gene was constructed and transferred to tobacco, five transgenic plants were obtained. Among them, the content of total flavonoids in leaves of four tobacco plants increased to different degrees compared with wild type control, and the highest line increased by 26.8%. The study indicated that AgF3H gene may play an important role in the biosynthesis of flavonoids in A. grossedentata and the results lay a foundation for further exploring the molecular mechanism of the formation of high flavonoids content in A. grossedentata.