In this paper, frozen steamed bread was used as the object to study the effect of frozen storage on the protein characteristics in a steamed bread system by measuring the changes in water holding capacity, oil holding capacity, emulsifying capacity, soluble protein content, surface hydrophobicity, secondary structure and molecular weights of gluten proteins in frozen steamed bread. The results showed that with an extension of frozen storage time, the disulfide bond (S-S) and total content of sulfydryl (-SH) decreased as a whole, and the molecular weights of steamed bread proteins were mainly distributed between 22 and 31 kDa, which were composed of a mixture of gliadin and low-molecular-weight glutenin subunits. With a decrease in frozen storage temperature, the soluble protein contents and protein water holding capacities of steamed bread first increased and then decreased, and the trend of the surface hydrophobicity was opposite. The frozen storage temperature had a significant effect on the protein properties of the steamed bread system. The changes in protein emulsification, oil holding capacity and soluble protein content were stable at -24 °C, which was suitable for long-term frozen storage.