Objective To optimize the extraction process of polyphenols from Clausena anisum-olens kernel, and to analyse their in vitro antioxidant and antibacterial activities.Methods With ultrasonic temperature, power, time, as well as solid-liquid ratio and ethanol volume fraction as experimental factors, the response surface method is employed to optimize the ultrasonic extraction process of polyphenols from C. anisum-olens kernel. Furthermore, the in vitro antioxidant activity of the polyphenols is investigated via free radical scavenging assays, and the antibacterial activity is determined using the well diffusion method.Results The optimal extraction process is an ultrasonic power of 94 W, an ultrasonic time of 17 min, a solid-liquid ratio of 1∶38 (g/mL), and an ethanol volume fraction of 70%. Under these conditions, the polyphenol yield from the C. anisum-olens kernel is (28.53±0.13) mg/g. The DPPH and ABTS⁺ free radical scavenging abilities of polyphenols exceed those of vitamin C, with half maximal inhibitory concentration (IC₅₀) for hydroxyl, DPPH, and ABTS⁺ free radicals being 0.37 mg/mL, 29.75 μg/mL, and 6.02 μg/mL, respectively. The minimum inhibitory concentration (MIC) for Escherichia coli and Staphylococcus aureus is 5.63 mg/mL, while the MIC for Bacillus proteus and Pseudomonas aeruginosa is 2.81 mg/mL.Conclusion With the optimized extraction process, a higher yield of polyphenols from the C. anisum-olens kernel can be obtained. Additionally, with potent antioxidant and antibacterial activities, these polyphenols demonstrate a promising application prospect in natural antioxidant and antibacterial agent development.