Abstract: In this study, the inhibitory effect of rape bee pollen polyphenols on pancreatic lipase was measured by enzyme kinetics method, and the interaction between pancreatic lipase and rape bee pollen polyphenol extracts was analyzed by the ultravilolet, fluorescence spectra and synchronous fluorescence spectra. The results showed that rape bee pollen polyphenols had a certain inhibitory effect on the activity of pancreatic lipase, and the half maximal inhibitory concentration (IC₅₀) was 1.670±0.045 mg/mL. The inhibitory type was a mixed inhibition of reversible inhibition. Fluorescence spectra showed that the maximal fluorescence intensity of pancreatic lipase gradually decreased with the increasing of rape bee pollen polyphenols concentration, and the maximal fluorescence peak had a redshift from 341 nm to 349 nm. Synchronous fluorescence spectra showed that the hydrophobicity inside the enzyme decreased and the stretch of the peptide chain increased when polyphenols bind to pancreatic lipase. The fluorescence quenching mechanism showed that the quenching constant decreased from 0.1000 to 0.0743 with increasing temperature, and the binding process was static quenching with about one binding site. In conclusion, the rape bee pollen polyphenols could inhibit the pancreatic lipase activity by changing the enzyme conformation, which provided a certain theoretical basis for the mechanism of bee pollen lipid reduction.