Abstract: This study aimed to investigate the enzymatic hydrolysis effects of papain, alkaline protease, neutral protease, alkaline protease TG enzyme combination, and papain alkaline protease combination on gluten protein and their impact on its structure. The degree of hydrolysis, trichloroacetic acid nitrogen solubility index (TCA-NSI), and molecular weight distribution were used as evaluation indicators to the enzymatic hydrolysis. The surface hydrophobicity, free thiol groups, and protein microstructure were determined using Fourier transform infrared spectroscopy, intrinsic fluorescence spectroscopy, UV spectroscopy, and other methods, and the structural changes of enzymatic hydrolysis products were systematically characterized. The results showed that enzymatic hydrolysis significantly increased the degree of hydrolysis of wheat gluten protein. The increase in surface hydrophobicity and free thiol content indicated significant changes in the protein's tertiary structure. Secondary structure analysis showed that in wheat gluten protein, the β-folding structure was transformed into irregular curls. The comparison of molecular weight distribution before and after enzymatic hydrolysis showed that the papain alkaline protease combination could more effectively hydrolyze wheat soluble protein. In addition, changes in the microstructure indicated that enzymatic hydrolysis altered the conformation of protein molecules. Therefore, this study provides theoretical support for the study of structural changes in wheat gluten after protein hydrolysis, and provides scientific basis for the development of high solubility and high functionality gluten protein products.