Objective To explore the interaction mechanism between diethyl phthalate (DEP) and ovalbumin (OVA) using multispectral analysis and molecular docking techniques.Methods The Stern-Volmer equation is applied to study the fluorescence quenching mechanism of OVA by DEP, as well as to determine the binding constants and the number of binding sites between them. Thermodynamic parameters are used to identify the primary types of interaction forces. Based on F?rster's non-radiative energy transfer theory, the energy transfer efficiency and binding distance between DEP and OVA are calculated. Molecular docking simulations are used to investigate the interaction forces between DEP and OVA.Results DEP can statically quench the intrinsic fluorescence of OVA. At temperatures ranging from 25 to 45 ℃, the binding constant (K) decreases from 2.631×10⁸ L/mol to 1.072×10⁸ L/mol, with 1~2 major binding sites identified between DEP and OVA. Thermodynamic analysis suggests that the interaction occurs spontaneously through electrostatic forces. Fluorescence resonance energy transfer parameters (J=1.316×10^-13 cm³·L/mol, R₀=3.665 nm, E=0.735, r=3.091 nm) confirms the strong binding. UV-Vis spectroscopy shows that DEP and OVA form a ground-state complex. Molecular docking simulations demonstrate that DEP binds to OVA through hydrogen bonds, van der Waals forces, and hydrophobic interactions, resulting in a stable complex structure.Conclusion DEP can statically quench the intrinsic fluorescence of OVA. The two molecules spontaneously form a stable ground-state complex with 1~2 binding sites, a binding distance of 3.091 nm, and an energy transfer efficiency of 0.735. The stability of the complex structure is primarily maintained by hydrogen bonds, van der Waals forces, and hydrophobic interactions.