Spectroscopy and molecular simulation technologies investigated The interaction mechanism between ellagic acid (EA) and urolithin A~D (UA~D) with HSA, which helped explore its pharmacotoxicity and efficacy. The results indicated that EA and UA~D could bind with HSA at a molar ratio of 1∶1 and quench its fluorescence via a static mechanism. The binding of UA and UC with HSA was exothermic and driven by hydrogen bonding and van der Waals force. The binding of EA and UD with HSA was endothermic and driven by hydrophobic interaction. The three-dimensional fluorescence spectrum analysis exhibited that the addition of EA and UC, UD, UA, and UB enhanced the hydrophilicity and hydrophobicity of tyrosine and tryptophan microenvironments of HSA, respectively. The molecular simulation analysis showed that EA and UA~D formed hydrogen bonds with active amino acid residues Lys436, Asp187, Lys432, Arg485, Leu430, Leu4, Ile388 and Tyr411, and formed hydrophobic interaction with active amino acid residues Ala191, Val456, Lys199 and Trp214, which proved that they were majorly bound to HSA by hydrogen bonds and van der Waalsforce, and then screened glycation sites and inhibited HSA glycation. This study could provide a theoretical basis for developing EA and UA~D as non-enzyme glycation inhibitors to treat diabetic complications.