Objective: The present study aimed to optimize the extraction process of polyphenols from Trifolium pratense Linn and evaluate the antioxidant activity in vitro. Methods: Ultrasonic-assisted extraction of total polyphenols from stems, leaves, and flowers was firstly performed, and the parts with higher polyphenol content were selected for further investigation. Then the response surface methodology was used to optimize the extraction process, and its antioxidant activity was evaluated by scavenging rate of hydroxyl radical and superoxide anion radical. Results: The results showed that the content of polyphenols in the leaves is relatively high. The optimal extraction process is as follows: the ratio of solid to liquid is 1∶79 （g/mL）, the extraction time is 23 min, the volume fraction of ethanol is 38%, the ultrasonic power is 800 W, and the ultrasonic temperature is 50 ℃; the yield of polyphenols is 2.31%, which is close to the predicted return of 2.26%, and the relative standard deviation is 2.16% （n=5）. The IC₅₀ to hydroxyl radical and superoxide anion radical was 0.419 mg/mL and 0.428 mg/mL, respectively. Conclusion: The results indicate that this extraction method is reliable and effective in extracting phenolic compounds from the leaves of Trifolium pretense Linn, and the extracts have good antioxidant activity.