Ultrasound pretreatment of wheat germ protein can increase the activity of hypoglycemic peptides prepared from enzymatic hydrolysis, and it is of great theoretical and practical significance to clarify the effect of ultrasonic wave on the structure of wheat germ protein. In this study, wheat germ protein was pretreated by ultrasonic wave with different time, and the structure of the protein was analyzed by fluorescence and Raman spectroscopy, and then the structure of lyophilized protein was analyzed by IR spectroscopy. The results showed that ultrasonic pretreatment influenced the enzymatic hydrolysis of hypoglycemic peptides, and the activity raised to the highest at 30 min. The fluorescence spectroscopy indicated that the pretreatment could induce the transferring of tryptophan residues from hydrophobic to hydrophilic regions in wheat germ protein. However, the protein structure was not found significant damage. Raman spectroscopy showed that protein structure was strongly affected at 30 min, and the content of β-sheet decreased to the lowest. Infrared spectroscopy analysis showed that protein structure changed with time, and the maximum change was at 35 min. Moreover, the α-helix content decreased significantly, while the random-coil content increased significantly. Raman spectroscopy and infrared spectroscopy provided more information than the fluorescence. Raman spectroscopy provided more accurate substrate structure in water-soluble enzyme catalytic system, and this showed more instructive for the protein enzymatic reactions.