Tetrastigma hemsleyanum Diels et Gilg, which is called “plant antibiotic”, possesses attractive effects including anti-inflammatory, anti-tumor, and antioxidant, and its efficacy is attributed mainly from flavonoids. However, the flavonoid composition and the expression of flavonoid-related genes are rarely studied in T. hemsleyanum. In this study, differences in flavonoids were observed between leaves and root tubers in T. hemsleyanum. A total of 22 flavonoids were detected, among them, luteolin, luteoloside, hesperidin, rutin, lonicerin, baicalin, and apigenin-7-O-glycoside were only contained in leaves, while kaempferol was only contained in root tubers. Concentrations of vitexin, orientin, lonicerin, rutin, isoquercitrin, luteolin, luteoloside, hesperidin, and naringin in leaves were higher than those in root tubers; concentrations of astragalin and kaempferol in root tubers were higher than those in leaves. Transcriptome analysis revealed differentially expressed genes (DEGs) in the two different medicinal portions were enriched in Photosynthesis-antenna proteins. Pearson correlation analysis indicated that the expression of chalcone isomerase (CHI) and UDP-glycose flavonoid glycosyltransferase (UFGT) was highly correlated with the concentrations of most flavonoids. The study revealed that DEGs in Photosynthesis-antenna protein essentially contributed to the difference in flavonoids in leaves and root tubers. CHI and UFGT were the potential key genes of accumulation of most flavonoids in T. hemsleyanum.