In this study, the seeds of Prunus humilis were used to extract amygdalin from the kernels by using ultrasound-assisted ethanol , and HPLC was used for detection. The amygdalin extraction rate was used as the detection index, and the ethanol concentration and the liquid-to-material ratio, extraction temperature, ultrasonic time and ultrasonic power were tested by single factor test, and then the relationships between each factor and extraction rate were analyzed. Thereafter, based on the two-level, five-factor experiment and response surface method, ultrasonic extraction process were optimized to obtain the extraction of Eucalyptus seeds. The optimum conditions of amygdalin included liquid to material ratio 13∶1 (mL/g), ethanol concentration 79%, extraction at 70 ℃, ultrasonic for 40 min, and ultrasonic power 192 W, and the final yield was 6.01%.