Objective:This study aimed to optimize the optimal extraction process of total flavonoids from the root of Clausena anisum-olens and determine the yield and antibacterial effect of total flavonoids from different producing areas. Methods:The total flavonoids were extracted by ultrasonic method from the roots of C. anisum-olens. Through single factor and orthogonal test, the effect of ultrasonic assisted extraction conditions on the extraction rate of total flavonoids from the root of C. anisum-olens was investigated, and the extraction and antibacterial test of total flavonoids from various producing areas were carried out. Results: The optimum extraction process of total flavonoids from C. anisum-olens was as follows: temperature 70 ℃, ultrasonic power 50 W, ethanol concentration 90%, solid-liquid ratio 1∶16 (g/mL), extraction time 40 min. Under the control of these conditions, the extraction rate of total flavonoids was 3.361%, and the yield of total flavonoids in the root bark of C. anisum-olens in Chongzuo city was the highest, up to 4.781%; the total flavonoids of C. anisum-olens had strong inhibitory effects on Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, borer killing bacteria and Escherichia coli, indicating that it had broad-spectrum antibacterial activity. Conclusion: Ultrasonic method was suitable for the extraction of flavonoids from the root of C. anisum-olens. The yield of flavonoids from different producing areas varied greatly, and the total flavonoids had broad spectrum antibacterial activity.