Abstract: This study delved into the inhibitory activity and underlying mechanism of a combination of three flavonoids against pancreatic lipase (PL), aiming to furnish a robust theoretical foundation for the development of natural anti-obesity pharmaceuticals. The optimal proportion of these three flavonoids for combination was determined through the use of a combined equivalent line graph. Subsequently, ultraviolet (UV) spectroscopy and fluorescence spectroscopy were employed to explore the binding reactions and mechanisms between genistein (Gen), chrysin (Chr), naringenin (Nar), and PL. To comprehensively elucidate the impact of these flavonoids on the structure and properties of PL, synchronous fluorescence, infrared spectroscopy, differential scanning calorimetry, and particle size analysis were simultaneously utilised to characterize the formed complexes from multiple perspectives. The results showed that when the molar ratio of the three flavonoids was 4:1, the combined inhibitory effect was the most pronounced, exhibiting a synergistic action. Both endogenous fluorescence and UV spectrum demonstrated that the quenching of PL by Gen-Chr, Gen-Nar, and Nar-Chr was static quenching. The corresponding binding constants K_a were calculated to be 1.63×10⁵ L/mol, 14.64×10⁵ L/mol, and 7.07×10⁵ L/mol, respectively. Thermodynamic parameter analysis indicated that hydrophobic interaction was the dominat force in the Gen-Chr-PL complex, electrostatic attraction played a predominant role in the Gen-Nar-PL complex, and hydrogen bonding along with van der Waals force were the main driving forces in the Nar-Chr-PL complex. The results from synchronous fluorescence and particle size results indicated that as the concentration of the inhibitors increased, the hydrophobicity of the complexes decreased. Infrared spectroscopy results indicated that the inhibitors altered the secondary structure of PL. Differential scanning calorimetry (DSC) results showed that the thermal stability of PL diminished after the formation of complexes with the three compounds. Collectively, these results suggest that the pairwise combinations of Gen, Chr, and Nar exert inhibitory effects on PL activity and can significantly alter the structure and properties of PL, providing new insights for the development of novel PL inhibitors.