The feasibility of producting flavonoids from callus of Tetrastigma hemsleyanum was investigated through callus induction, proliferation, differentiation and determination of flavonoids.The leaves of sterile plantlet, leaves and stems of wild plants were used as explants to induce calluses; The root tuber, the leaves and calluses were selected for the determination of flavonoids. With ethanol as the solvent, the total flavonoids were extracted by ultrasonic and determined by spectrophotometry at 500 nm after stained with NaNO2-Al(NO3) 3.The optimum medium where the calluses were induced was 2/3MS +2.0 mg/L 6-BA +2.0 mg/L NAA; MS +2.0 mg/L 6-BA +2.0 mg/L NAA was the optimum for callus proliferation; for callus root differentiation, the optimum medium was 1/2MS +1.0 mg/L 6-BA +1.0 mg/L NAA. The content of total flavonoids was 31.121 mg/g in root tuber, 12.830 mg/g in leaves while it was up to 18.088 mg/g in calluses.The calluses had a high level of total flavonoids, it could produce flavonoids through the calluses induced by Tetrastigma hemsleyanum in a large scale. In that case, the pressing requirement in medical market will be solved.