The inhibitory effect of Lycium barbarum leaves flavonoids on pancreatic lipase activity was explored by enzyme kinetics. The interaction mechanism between them was studied by ultraviolet， fluorescence and infrared spectroscopy. The results demonstrated that the flavonoids had an inhibitory effect on pancreatic lipase activity with a half-inhibitory concentration (IC50) of (0.910±0.008) mg/mL， inhibition type was reversible and non-competitive inhibition， and the constant of inhibition was 6.04 mg/mL. Ultraviolet and fluorescence spectra revealed that the flavonoids could result in the exposure of aromatic amino acids of pancrelipase， the change of microenvironment and enzyme conformation， and quenching of endogenous fluorescence. The quenching type was mainly dynamic quenching mixed quenching. Thermodynamic parameters showed that the interaction force between them was mainly hydrophobic force. Infrared spectroscopy revealed that the effect of the flavonoids on the secondary structure of enzyme protein was mainly as follows: the structure of the compound became orderly， and the β-sheet was transformed to α-helix. The flavonoids could change the structure and microenvironment of pancrelipase and then inhibit its activity.