In this paper, the interaction between fulvic acid (FA) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, ultraviolet spectroscopy, circular dichroism, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and molecular docking simulation in the simulated physiological environment. In the fluorescence spectroscopy study, the dynamic fluorescence quenching rate constant K_q and quenching constant at 298, 303 and 308 K are calculated by the Stern-Volmer equation, which proves that the quenching process of the interaction between BSA and FA is static quenching. At the same time, according to the calculated binding sites n, the interaction ratio between FA and BSA is 1:1. The thermodynamic parameters at three temperatures are calculated by static quenching double logarithm equation, enthalpy change ΔH<0, entropy change ΔS<0, it is concluded that the main interaction force between FA and BSA is hydrogen bond and van der Waals force, ΔG<0, indicating that the interaction process is spontaneous. Based on Förster's dipole-dipole non-radiative energy transfer theory, the binding distance rang 6.340 nm is calculated, indicating a non-radiative energy transfer between BSA and FA. The molecular docking simulation results show that the binding force between FA and BSA residues is hydrogen bond and van der Waals force, and there is a hydrophobic force between them. The interaction of multiple forces makes FA and BSA combine stably. Through the UV-Vis absorption spectrum analysis of the interaction between FA and BSA, it is found that the maximum absorption peak of BSA has an obvious red-shift, indicating that FA changes the secondary structure of BSA. By studying the synchronous fluorescence spectrum of the interaction between FA and BSA, it was found that FA enhanced the polarity of the microenvironment around the tryptophan (Trp) residue in BSA, weakened its hydrophobicity and enhanced its hydrophilicity, which changed the protein conformation of BSA to a certain extent. Through the study of the three-dimensional fluorescence spectrum of the interaction between FA and BSA, the maximum emission wavelengths of peak 1 (peak 1) and peak 2 (peak 2) were red-shifted, which proved that FA interacted with BSA. FA increased the polarity of the environment around BSA, decreased its hydrophobicity, increased its hydrophilicity, and changed the protein conformation of BSA. Finally, circular dichroism was used for analysis, and the software was used to calculate that under the experimental interaction system, α-helix (α-Helix) decreased by 2.3%, β-sheet increased by 7.7%, β-Turn increased by 0.6%, and irregular structure (Random coil) content decreased by 1.2%. The content of β-sheet increased most obviously, which strongly indicated that FA changed the structure of BSA.