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Background: The effective extractives of Tetrastigma hemsleyanum (T. hemsleyanum) are useful, but this plant was not fully utilized in rural China. In this study, flavonoids from the aerial parts of T. hemsleyanum were extracted, and the purification method and the antioxidant capability were explored. Purpose: This study is to explore the optimized purification method and the biological capability of the total flavonoids extracted from T. hemsleyanum. Materials and Methods: The optimal extraction conditions were determined by single-factor and Box-Behnken design experiments. The antioxidant activities were assessed by analyzing their capability to scavenge 2,2-diphenyl-1-picrylhydrazyl and OH free radicals. Results and Discussion: The optimal conditions for the extraction of the total flavonoids were as follows: ethanol volume fraction 60%, liquid-to-material ratios 35:1 mL/g, temperature 45℃, and time 85 min. The extraction rate of the total flavonoids was (3.59 ± 0.03)%. NKA-9 is an ideal macroporous resin for purification. The optimized purification process conditions of flavonoids were mass concentration of adsorption solution 1.50 mg/mL, pH value of adsorption solution 4.0, sample loading flow rate 2 BV/h, ethanol volume fraction in desorption process 60%, desorption flow rate 2 BV/h and elution volume 4 BV. After crude extracts were purified by NKA-9 macroporous resin, the content of the total flavonoids enhanced from (21.76 ± 0.18)% to (58.92 ± 0.22)%. The results of the experiments for antioxidant activity showed that the half maximal inhibitory concentration (IC50) values of the purified flavonoids extracted were 33.1and 42.7 µg/mL, respectively. Conclusion: The results of this study indicated that the flavonoids in T. hemsleyanum have good antioxidant activity in vitro and deserve exploring in further pharmacological studies. [ABSTRACT FROM AUTHOR]