Abstract: To explore the optimum extraction process of flavonoids from Portulaca oleracea L. by methanol leaching-ultrasonic assistance and evaluate its antioxidant and anti-cancer activities. Based on single factor experiment, methanol concentration, extraction temperature, solid- liquid ratio and ultrasonic power were enrolled in this experiment, and the extracting ratio of total flavonoids was used to evaluate the extraction process. The extraction process of total flavonoids from P. oleracea L. was optimized by orthogonal experiment. Meanwhile, the antioxidant activity of flavonoids was assessed by scavenging assays of DPPH· and ·OH. The viability of human lung cell line A549 was detected by MTT assay. The optimal extraction process was as follows: liquid-solid ratio of 1:50, water bath temperature of 80℃, ultrasonic power of 200 W, 90% methanol as extraction solvent, extraction time of 30 min, extraction of 2 times. The extraction rate of total flavonoids from P. oleracea L. was 6.37% under the optimal condition. The scavenging rate of DPPH· was 89.80% at 4.0 mg/mL, while the scavenging rate of ·OH was 86.07% at 20 mg/mL. The inhibition rate of cell A549 proliferation was 74.62% at 420 μg/mL, which had no significant difference from that of the positive drug group. The flavonoids from P. oleracea L. are extracted efficiently by the above optimized technology and the total flavonoids exhibit antioxidant and antitumor activities.