In this paper, wheat flour was irradiated at different doses (0, 5, 10, 15, 20 and 25 kGy) to investigate the effects of 60Co-γirradiation on the structural and functional properties of its gluten. The disulfide bond was broken by irradiation, and the content of the free sulfhydryl group increased. The results of Fourier infrared spectroscopy showed that the secondary structure of gluten protein was rearranged with decrease in a-helix content and an increase in β-sheet content. Raman spectroscopy and surface hydrophobicity showed that the microenvironment of aromatic amino acid side chains changed. Irradiation treatment resulted in the tryptophan and tyrosine residues tending more toward the exposed state, and the surface hydrophobicity increased. Meanwhile, irradiation increased the solubility, water and oil holding, and emulsifying properties of gluten proteins, and the maximum value reached at 15 kGy（solubility is 0.46 mg/mL, water holding 3.13 g/g, oil holding 4.33 g/g, emulsification 6.669 m2/g）. In conclusion, the proper dose of gamma-irradiation can change the secondary and tertiary structure of gluten, and improve its functional properties such as solubility, water retention and emulsification, which is conducive to expanding the application space of gluten protein and providing theoretical references for industrial applications.