In this study, the natural antioxidants myricetin, morin, capsaicin and betaine were selected as the research objects, and the interaction between several antioxidants, DPPH free radical and human albumin was studied by synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy. Results showed that capsaicin, betaine, VC do not have quench effects with human serum albumin, and myricetin, morin and DPPH have quench effects with human albumin. The reactions are static quenching caused by stable complexes, combining hydrophobic interaction with HSA.,The binding sites are 1, the main binding sites are near the tryptophan groups, and DPPH and human serum albumin’s quenching process changes the structure of human serum protein hydrophobicity, causes protein conformation changes, and the interaction between myricetin, morin and human albumin does notcause its conformation changes. Fluorescence spectroscopy were used to study the inhibitory effect of several antioxidants on DPPH-induced direct damage to human serum albumin. The inhibition rate of myricetin, morin, capsaicin, betaine and VC to DPPH damage HSA was 25%, 18.30%, 85.38%, 4.02% and 84.58%. Capsaicin inhibits the damage of human serum albumin by inhibiting the action of DPPH. The binary system reaction results showed that myricetin reacted with Morin ternary system to form competitive binding sites with DPPH, and Myricetin and Morin inhibit DPPH damage to human serum albumin by occupying binding sites, while Betaine can neither occupy the binding sites nor scavenge free radicals, so the inhibition ability is the weakest. The results showed that the inhibitory ability of several natural antioxidants is closely related to the main functional groups in the molecular structure.