Abstract: To investigate the effect of oleic acid on the interaction mechanism between gluten protein and rutin, measurements were conducted using UV absorption spectroscopy, fluorescence quenching spectroscopy, synchronous fluorescence spectroscopy, and three-dimensional fluorescence spectroscopy to determine the quenching type, binding constant, binding site number, binding force type, and binding distance. At present, interaction of gluten proteins with polyphenols has been increasingly studied in recent years, but little has been done to address the effect of fatty acid addition on the interaction of gluten proteins with polyphenol complexes. The results showed that oleic acid could alter the microenvironment surrounding gluten protein, reducing the fluorescence quenching rate of rutin on gluten protein. The quenching mode of rutin on gluten was a combination of dynamic and static quenching, with quenching being dominant, and oleic acid did not change this quenching mode. Oleic acid reduced the binding constant and binding sites between rutin and glutenin protein, weakened their binding force, and altered the interaction forces between gluten protein and rutin. Oleic acid could from new complexes with the glutenin protein-rutin complex system, causing changes in the protein structure of gluten protein through synergistic and non-competitive binding, which inhibited the binding between gluten protein and rutin. This study reveals the mechanism of oleic acid’s influence on the interaction between gluten protein and rutin at the molecular level, providing a theoretical basis for regulating the functional properties of food.