The present invention provides a method for preparing a gluten ACE inhibitory peptide through multi-mode ultrasonic wave strengthened enzymolysis, and relates to the technical field of deep processing of agricultural products. According to the method, an ultrasonic pretreatment is applied on a gluten suspension, wherein the sequential work mode frequency combination is 20-35 kHz, the sequential time is 5 s and 5 s, the synchronous work mode frequency combination is 20-28 kHz, the pulse time is 10 s, the interval time is 5 s, the ultrasonic power per unit volume is 200 W / L, and the pretreatment time is 15 min; alkaline protease is added to carry out enzymolysis after the pretreatment, wherein the enzymolysis time is 30 min; and the enzyme is inactivated for 10 min through a 100 DEG C boiling water bath, centrifugation is performed for 10 min under 10000 g, and the supernatant is collected so as to obtain the extraction liquid of the gluten ACE inhibitory peptide. According to the present invention, the gluten suspension is subjected to the multi-mode ultrasonic wave pretreatment so as to increase the enzymolysis efficiency, wherein the ACE inhibition rate activity of the hydrolysate is increased by 16.4-25.4% compared with the traditional enzymolysis method, and the ACE inhibition rate activity of the hydrolysate is increased by 6.0-13.7% compared with the counterflow type single-frequency ultrasound.