In this study, we evaluated the inhibitory effect of spinosin (SP) on the glycation of β-lactoglobulin (β-Lg) using fluorescence spectroscopy, ultraviolet visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), thioflavin T (ThT) staining, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Fluorescence spectroscopic results showed that SP effectively inhibited the formation of advanced glycosylation end products (AGEs), stabilized the conformational structure of glycated β-Lg, and altered the microenvironment near Trp and Tyr residues. UV-Vis spectroscopy showed that glycation changed the structure of β-Lg, and SP inhibited the production of AGEs by slowing down the late stage of the Maillard reaction. FTIR spectral analysis showed the involvement of hydrogen bonding in the formation of SP-β-Lg complex. The results of ThT staining showed that SP reduced β-amyloid deposition by attenuating protein misfolding. SDS-PAGE analysis showed inhibition of SP-induced cross-linking of glycated β-Lg. These results demonstrate that SP holds promise as an anti-glycation agent in food systems, with the potential to reduce the formation of harmful glycation products.