In this study, the interaction between fulvic acid (FA) and cellulase (CEL) and its impact on CEL conformation were systematically investigated using fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, UV-VIS absorption spectroscopy, circular dichroism, infrared spectroscopy, and molecular docking simulations. Fluorescence quenching analysis revealed that the fluorescence intensity of FA decreased in a concentration-dependent manner with increasing CEL concentration. Moreover, the quenching constant decreased with rising temperature, indicating FA's significant static quenching effect on CEL fluorescence. Thermodynamic analysis demonstrated that the interaction between FA and CEL is primarily driven by van der Waals forces and hydrogen bonding, rendering the reaction thermodynamically spontaneous. Förster resonance energy transfer theory suggests potential non-radiative energy transfer between FA and CEL. Ultraviolet-visible absorption and synchronous fluorescence spectra further confirmed the static quenching mechanism and indicated changes in FA's structural density and amino acid residues' microenvironment. Circular dichroism analysis revealed that the binding site of FA and CEL is located in the hydrophobic cavity near the tryptophan residue, leading to reduced hydrophobicity in CEL's secondary structure and an extended peptide chain. This finding corroborates the interaction between FA and CEL and the influence of FA on CEL's secondary structure. Molecular docking studies also showed that the interaction between FA and CEL is predominantly governed by van der Waals forces and hydrogen bonds, achieving stable binding through these interactions, which aligns with the thermodynamic results. Infrared spectroscopy results further support this conclusion.