Abstract: To optimize the process of ultrasound-assisted enzymatic extraction of total flavonoids from the roots of Houttuynia cordata, the appropriate liquid to material ratio, particle size, ethanol concentration, enzymatic digestion time, and enzyme addition was screened out by single-factor experiments, and the optimal conditions for the extraction of the flavonoids from Houttuynia cordata were optimized by Box-Behnken response surface. Meanwhile, the antioxidant activity of flavonoids was investigated. The results showed that the optimum extraction process was liquid to material ratio of 28:1 mL/g, particle size of <250 μm, ethanol concentration of 54%, and enzymatic digestion time of 22 min. The extraction amount of flavonoids was 22.197 mg/g under the optimized conditions, and the purity reached 26.63% by concentration under reduced pressure. The obtained flavonoids showed good scavenging ability for DPPH·, ·OH, and ABTS⁺· with EC₅₀ values of 0.097, 2.250, and 0.384 mg/mL, respectively. The results of the study would provide a theoretical basis for the extraction of total flavonoids from the roots of Houttuynia cordata and a reference for further realization of the utilization of Houttuynia cordata.