The antiglycation effects of three structurally different phytosterols (PS) including stigmasterol, β-sitosterol, and γ-oryzanol on bovine serum albumin (BSA) were deeply studied in a BSA-glucose model by measuring the glycoxidation-based products, SDS-PAGE intensity, free lysine, and their fluorescence microscopy clicks. For the first time, the underlying mechanisms of the antiglycation effects of PS were wholly elucidated by measuring their interaction ability with BSA and their antiradical activity during the glycation reactions. The results showed that PS could partially inhibit the formation of advance glycation end products, block some of the lysyl residues of BSA (Lys127, 357, 434, and 524), prevent the glucose-BAS bonding, and their disaggregation effects on the glycated BSA. Throughout the underlying mechanism behind the antiglycation activity, PS were found to structurally quench the fluorescence intensity .