Abstract: The purpose of this experiment was to optimize the extraction process of total flavonoids from Scutellaria barbata and to evaluate the antioxidant and antitumor activity of total flavonoids. A single factor experiment combined with a response surface Box-Behnken design was used to study the extraction process of total flavonoids from Scutellaria barbata. The effects of extraction time, material-to-liquid ratio, extraction temperature and volume fraction of ethanol on the yield of flavonoids were investigated to arrive at the optimal extraction process of total flavonoids from Scutellaria barbata. The antioxidant activity of total flavonoids from Scutellaria barbata was detected by DPPH and ABTS methods, and the effect of purified products on the proliferation of NCI-H1299, HepG2, MHCC-97H and HuH-7 cells were analyzed using the MTT assay. The results showed that the optimum extraction conditions of total flavonoids from Scutellaria barbata were as follows: Extraction time 93 min, ratio of material to liquid 1:41 (g/mL), extraction temperature 68 ℃, volume fraction of ethanol 75%. Under these conditions, the yield of total flavonoids was 26.46 mg/g. The results of antioxidant experiments showed that the purified total flavonoids of Scutellaria barbata had good scavenging effects on DPPH and ABTS⁺ radicals, with IC₅₀ values of 25.41 and 70.41 μg/mL, respectively. The results of anti-tumor experiments showed that the purified total flavonoids of Scutellaria barbata at different mass concentrations within a certain concentration range had certain inhibitory effects on the proliferation of tumor cells, with the IC₅₀ values of NCI-H1299, HepG2, MHCC-97H and HuH-7 cells were 168.6, 330.5, 269.2 and 335.8 μg/mL, respectively. In summary, it could be concluded that the method was stable and feasible, reproducible, and could effectively extract the total flavonoid components from Scutellaria barbata, and the purified Scutellaria barbata total flavonoids had good antioxidant and antitumor activities, which could be used for the development of functional foods or pharmaceuticals.