Abstract: To investigate the interaction mechanisms between gluten protein, kaempferol, and resveratrol, fluorescence spectroscopy, ultraviolet-visible (UV-Visible) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, and molecular docking were applied. Molecular interaction information such as fluorescence quenching mechanism, interaction force type, and changes in protein secondary structure were determined. The results showed that kaempferol mainly quenched the intrinsic fluorescence of gluten protein through static quenching, while resveratrol quenched gluten protein through a combination of dynamic and static quenching. Compared to resveratrol, kaempferol bound gluten protein with a stronger affinity. Thermodynamic studies had shown that the binding of two polyphenols to gluten protein was mainly driven by hydrogen bonding and van der Waals forces. The binding process was a spontaneous exothermic reaction. Kaempferol’s binding site to gluten protein was closer to tryptophan residues, while resveratrol’s binding site was closer to tyrosine residues. FT-IR spectroscopy analysis showed that kaempferol and resveratrol reduced the structural order and stability of gluten protein, increased the content of gluten protein β-sheet, and decreased the α-helix and antiparallel β-sheet. Molecular docking analysis indicated that gluten protein mainly interacts with kaempferol and resveratrol through hydrogen bonding and van der Waals forces. This study reveals the interaction mechanisms between kaempferol, resveratrol, and gluten protein, providing data support for the utilization and processing of gluten protein foods.