Quercetin has been reported to suppress protein glycation or the formation of advanced glycation end-products (AGEs), but the inhibition mechanism related to protein structure and glycation sites and the influence on physicochemical properties remain unclear. The aim of the current research was to investigate the mechanism of quercetin against glycation with BSA-fructose as model by spectroscopic and spectrometric techniques. Changes in physicochemical properties were evaluated by antioxidant activity and emulsifying properties. The results indicated that quercetin dose-dependently inhibited the glycation of BSA by attenuating the alteration of conformational structure and microenvironment induced by glycation. It could also suppress the cross-linking or aggregation of glycated BSA, which reflected in the decreased molecular weight determined by SDS-PAGE and MALDI-TOF. Nanoliquid chromatography coupled to Q-Exactive tandem mass spectrometry analysis revealed the mapping of 20, 23, 19, and 19 glycation sites in glycated BSA with 0, 0.5, 1.5, and 3.0 mM quercetin, respectively. Quercetin changed the glycation sites of BSA, but it could not reduce the number greatly. In addition, quercetin reduced the antioxidant ability and increased the emulsifying properties of BSA, while negligible efficiency was observed on the antioxidant activity and emulsifying activity index of glycated BSA.