The optimal operational and process parameters were determined for the enzymatic hydrolysis and ultrasound-assisted extraction (EHUE) method of flavonoids extracted from Taxus cuspidata branches and leaves (TCBL), and the biological activity of obtained flavonoids was evaluated. According to single factor and central composite design experiments, the optimum key experimental parameters for EHUE were that pectinase enzyme concentration was 0.10 mg·mL-1, enzymatic hydrolysis temperature was 48 °C, and enzymolysis time was 39 min. The yield of flavonoids from TCBL under the optimized conditions was 5.23% ± 0.18%. Four purified flavonoid compounds from TCBL extract were identified as 1) (E)-1-methoxy-2-O-(p-coumaroyl)-myo-inositol, 2) catechin, 3) epicatechin, and 4) quercetin-3-O-glucoside. Among the 4 compounds, compounds 2 and 3 showed higher antioxidant capacities, α-amylase, and α-glucosidase inhibitory activities. The statistical analysis showed that epicatechin and catechin were potent antioxidants and active agents for inhibiting type II diabetes. In addition, all 4 compounds exerted clear antitumor activity against MCF-7, Hela, and HepG2 cells. Especially, compound 4 had highest antitumor capacity against MCF-7 and Hela, while compound 1 was best at suppressing the proliferation of HepG2 cells.