The inhibition of Nelumbo nucifera leaf (NNL) extract and its fractions on the formation of advanced glycation end-products (AGEs) was evaluated with different models. Changes in the conformational structure of bovine serum albumin (BSA) were analyzed by fluorescence spectrometry. Effect of ethyl acetate fraction (EAF) on the glycation sites and glycation degree of BSA was investigated by Nano LC-Orbitrap-MS/MS due to its strongest inhibitory ability. Results indicated that EAF could effectively inhibit the formation of AGEs by scavenging reactive radicals and attenuating the conformational change of BSA induced by glycation. Phenolics and flavonoids were the major contributors. Eleven (11 lysines) and ten (9 lysines and 1 arginine) glycation sites were identified from glycated BSA with and without EAF, respectively. Addition of EAF could not reduce the number of glycation sites of BSA, but could significantly decrease the glycation degree of most glycated peptides and alter the location of glycation.