Fluorescence spectroscopy has the advantages of high sensitivity, strong selectivity, small sample amount, simple operation, and can provide a variety of physical parameters of intermolecular interaction. So it is widely used to study the interaction between small molecules and proteins. To elucidate the potential of isoquercetin suppressing the formation of (advanced glycation end products, AGEs) during thermal processing of high-protein foods, intrinsic fluorescence, synchronous fluorescence and three-dimensional fluorescence spectroscopic techniques were employed to evaluate the inhibition through α-lactalbumin (α-La)-fructose model, and the mechanism was elucidated from the perspective of intermolecular interactions. The results showed that isoquercetin significantly decreased the conformational change of α-La induced by glycosylation, resulting in the inhibition on AGEs formation. The inhibition rate reached up to 74.66% when the sample concentration was at 36.58 μmol·L^-1. As revealed by synchronous and three-dimensional fluorescence spectroscopy, isoquercetin could change the conformation of α-La, promote the exposure of tryptophan to a more hydrophilic environment, and increased the hydrophilicity of its micro-environment. Inhibition kinetics and thermodynamic analysis showed that isoquercetin quenched the intrinsic fluorescence of α-La by static mechanism, and bound with α-La in a molar ratio of 1:1 to form a stable complex. The formation of isoquercetin-α-La complexes is a spontaneous and endothermic progressing, van der Waals forces and hydrogen bonds are the main driving forces.