Abstract: To investigate the effect of combined lactic acid bacteria and yeast fermentation on the structure of prolamin in corn-flour dough, this study focused on prolamin from corn-wheat mixed flour. Four fermentation methods were employed: natural fermentation (NC), yeast fermentation (YC), Pediococcus pentosaceus PE5 fermentation (PC), and combined Pediococcus pentosaceus PE5 and yeast fermentation (PYC). The effects of combined versus single fermentation on prolamin structure were systematically compared using techniques such as SDS-PAGE gel electrophoresis, Fourier transform infrared spectroscopy, intrinsic fluorescence spectroscopy, thermal analysis, and particle size analysis. Results indicated that a more porous microstructure in the dough was induced by co-fermentation. Compared with YC, the proportions of α- prolamin, β- prolamin, and γ- prolamin were increased by co-fermentation by 1.17%, 1.45%, and 0.64% respectively, while ω- prolamin was decreased by 2.35%. Additionally, free sulfhydryl content was decreased by 30.62%, and disulfide bond content was increased by 36.6%. In terms of secondary structure, the relative content of α- helix was decreased by 11.6%, β- sheet was increased significantly by 4.5%, and random coil was increased significantly by 2.8%. Prolamin from co-fermentation exhibited the highest fluorescence intensity, thermal denaturation temperature, and enthalpy value. Compared with YC, the average particle size of prolamin in co-fermentation was increased from 411.9 nm to 453.8 nm, protein surface hydrophobicity was increased, and bromophenol blue binding rate was raised by 1.52 times. These findings suggested that the hydrolysis of high-molecular-weight prolamin in the dough can be more effectively promoted by co-fermentation, leading to more significant structural changes than single-strain fermentation. This provided a theoretical basis for the application of combined Pediococcus pentosaceus PE5 and yeast fermentation in corn flour products.