In this study, differently sized particles of wheat flour (from 52.36 μm to 108.89 μm) were obtained by adjusting the distance between the rolls (0.02, 0.04, 0.06, 0.08, and 0.1 mm) of a heart mill. Results showed that reducing the particle size significantly increased the damaged starch (DS) content. Uniaxial tensile measurement of dough showed that reducing the particle size of wheat flour can effectively increase the maximum tensile resistance, but the extensibility reaches the maximum in samples at medium particle diameter (78 and 66 μm). Additionally, the ratio of dynamic moduli (″/') decreased with a reducing particle size. The results of disulfide bond content, gluten microstructure, showed that finer flour granulation can strengthen the gluten network. The steamed bread (SB) making test showed that SB made from wheat flour of a smaller particle size had a significantly smaller specific volume than that made from a larger particle size. The texture profile analysis showed that with a decrease of wheat flour particle size, the hardness, chewiness of SB increased, the resilience decreased, and there was no significant difference in adhesiveness. Overall, the quality of SB made flour of medium particles (78 μm) is better.