This study systematically investigated the interaction mechanism between fulvic acid (FA) and β-lactoglobulin (BLG) using fluorescence spectroscopy, UV-vis absorption, circular dichroism, and molecular docking simulations. The results demonstrated that FA quenches BLG fluorescence through a static quenching mechanism with a binding constant (KA) of approximately 10 3 L/mol and about one binding site (n≈1) at 298 K. Thermodynamic analysis revealed the spontaneous nature of this interaction (ΔG < 0), primarily driven by van der Waals forces and hydrogen bonding (ΔH < 0, ΔS < 0). FRET analysis indicated an intermolecular distance of 3.083 nm, confirming non-radiative energy transfer. Structural characterization showed that FA binding induces conformational changes in BLG, particularly affecting the microenvironment of tryptophan residues. These findings provide valuable insights into the molecular interactions between FA and milk proteins, suggesting potential implications for FA’s biological applications and functional food development.