The structure changes of defatted wheat germ protein Isolate (DWGP) treated by ultrasonic was determined by FTIR and fluorescence spectra, and the effect of its structure changes on the high-efficient enzymatic hydrolysis was studied. Research showed that the efficiency of hydrolysate could be improved by ultrasonic treatment. Compared with control group, inhibitory activity of the hydrolysate was increased by 23.96% after the treatment of 600 W for 10 min. The fluorescence intensity of DWGP after ultrasonic treatment was found discovered to be changed. An appropriate ultrasonic treatment can unfold the protein molecule and make the chromogenic groups uncovered, which contribute to the acquirement of the higher-activity inhibitory peptide. The effects of various ultrasonic power and time on the secondary structure of DWGP were quantitatively determined via analysis of the amide Ⅰ changes of infrared spectra using curve fitting method. Content of β-sheet was decreased and β-turn was increased after ultrasonic treatment, which could be the main factor to make the prepared inhibitory peptides high efficient. The results provide a theoretical basis for the mechanism research of enzymatic hydrolysis of ultrasonic treated protein.