Abstract: Objective: To study the optimal ultrasound-assisted extraction technology of polysaccharide from Hippophae rhamnoides(seabuckthorn) leaves and its effect on CT-26 cells. Method: Firstly, the ultrasonic temperature, ultrasonic power, ultrasonic time and ultrasonic frequency were selected to carry out single factor experiment. According to the single factor results, the optimal ultrasonic assisted extraction process was studied. The antitumor activity of seabuckthorn leaves polysaccharide was detected by MTT method, flow cytometry, RT-PCR and Western blot method. Results: When the ultrasonic temperature was 70 ℃, the ultrasonic power was 400 W, the ultrasonic time was 30 min, and the ultrasonic frequency was 2 times, the polysaccharide yield of seabuckthorn leaves was the highest to 7.93%±0.07%. The factors were ranked as follows: Ultrasonic power>ultrasonic time>ultrasonic temperature>ultrasonic frequency. MTT and transmission electron microscope results showed that different concentrations of seabuckthorn leaves polysaccharide could significantly promote the apoptosis of CT-26 cells after 72 h treatment, and the best inhibitory effect was at the concentration of 2000 μg/mL; seabuckthorn leaves polysaccharide could block CT-26 cells in S phase, and significantly increase the expression of caspase-3 and Bax, and downregulate Bcl-2 expression to induce the apoptosis of CT-26 cells. Conclusion: The optimal extraction process of polysaccharides from seabuckthorn leaves was optimized and it could effectively inhibit the proliferation of CT-26 cells, providing scientific reference for the utilization of seabuckthorn resources.