Objective: This study aimed to evaluate the antibacterial activity of total flavonoids from rooibos (TFR) against E. coli and explore the application of rooibos resources in food field. Methods: Total flavonoids were extracted by ultrasonic-assisted ethanol extraction, and the extraction process was optimized by response surface test. The antibacterial effects of TFR on E. coli were analyzed by disk diffusion method, double dilution method, growth curve change and transmission electron microscope test. Results: The optimal extraction conditions were as follows: ethanol volume fraction 53%, solid-liquid ratio 1∶27 (g/mL), ultrasonic temperature 77 ℃, ultrasonic time 68 min. Under the control of these conditions, the extraction rate of total flavonoids was 2.46%. The inhibition zone diameter, minimum inhibitory concentration and minimum bactericidal concentration of total flavonoids against E. coli were (11.40±0.21) mm, 12.5 mg/mL and 25 mg/mL, respectively. The growth of E. coli disappeared in logarithmic phase, and the cell morphology was damaged, including cell deformation, collapse and cell fluid leakage. Conclusion: Under the control of the optimal extraction conditions, the extraction rate of TFR was higher, and it had good antibacterial effect on E. coli.