Total flavonoids were extracted from Cleome viscosa by ultrasound-assisted extraction. The optimum extraction conditions were investigated based on single factor and orthogonal tests. Meanwhile, the antioxidant activities of total flavonoids were evaluated by studying hydroxyl radical (·OH) scavenging assay, 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH·) scavenging assay and nitrite scavenging assay. The results showed that the extraction rate of total flavonoids of Cleome viscosa was (2.711±0.002)% under the following conditions: material-to-liquid ratio of 1:15 (g/mL), ethanol concentration of 50%, extraction power of 40 W, ultrasonic time of 50 min, extraction temperature of 50 ℃. Total flavonoids of Cleome viscosa exhibited obvious ·OH and nitrite scavenging capacity, and showed a higher scavenging capacity to DPPH·, and the corresponding maximum scavenging rates were (52.48±0.88)%, (95.58±0.28)% and (57.27±0.15)%, respectively. The results revealed that total flavonoids of Cleome viscosa had excellent antioxidant activities.