Abstract: To investigate the binding mechanism of the interaction between the natural bacteriostatic flavonoid polyphenol quercetin (Q) and ovalbumin (OVA) as well as the changes in the structure and bacteriostatic activity. In this paper, multispectroscopy and molecular docking techniques were employed to study the interaction mechanism of Q and OVA. Differential scanning calorimetry and scanning electron microscopy were used to characterise the thermodynamic properties and morphological changes before and after the interaction between Q and OVA. The inhibitory ability of Q-OVA was determined by the inhibition circle and inhibition rate. Results showed that the endogenous fluorescence of OVA could be effectively burst by Q, and its burst type was identified as static burst. A stable complex with binding site 1 was formed through their interaction, and the interaction process was found to be spontaneous, with hydrogen bonding and van der Waals force identified as the main intermolecular forces. The content of β-folding and α-helix in the secondary structure of OVA increased after the addition of Q, and the content of β-turns and random curls decreased. The intermolecular hydrogen bonding increased, and the OVA molecules extended. The molecular docking results explained that the binding site of Q and OVA was located in the large hydrophobic cavity of OVA, and Q could form hydrogen bonds with LYS386, THR280, and ASN391 on OVA. There were van der Waals forces with residues such as PHE276, and hydrophobic interactions with LEU279. After the complexation of Q with OVA, the thermal stability of the protein was decreased, and the microstructure was changed from a spherical structure with a smooth surface to a flat and dense lamellar structure. Compared with OVA, the solubility of the complex was decreased, while the hydrophobicity and turbidity of the surface were increased. The antioxidant property was enhanced, and the antimicrobial effect on S. aureus and E. coli was improved. The above results demonstrated that complexes formed through the interaction of Q and OVA exhibited good bacteriostatic effects. This study can provide theoretical guidance for the preparation of flavonoid-protein complexes with excellent antibacterial effects and their application in food processing.