A natural plant named vine (Ampelopsis grossedentata) has been proven to exsert various bioactivities due to its major component of dihydromyricetin (DMY), but there is little information on its hypolipidemic function. In this study, the inhibition behavior of DMY based on pancreatic lipase (PL) assay was investigated by ultraviolet spectroscopy followed by a series of multiple-spectroscopy measurements including fluorescence spectroscopy, synchronous fluorescence spectroscopy and 3D fluorescence spectroscopy as well as the DMY-PL interaction mechanism by molecular docking. The half inhibitory concentration (IC₅₀) of PL detected by UV spectroscopy was 2.6×10^-4 mol·L^-1, showing its satisfactory lipid-lowering capacity on PL. The calculation of the Lineweaver-Burk equation indicated their interaction type was competitive inhibition with the inhibition constant of 6×10^-4 mol·L^-1. The Stern-Volmer equation and static quenching double logarithmic formula analyzed the fluorescence spectra. The results suggested that DMY could significantly quench PL's self-luorescence and its fluorescence quenching constant K_SV was negatively sensitive to temperature, revealing that the fluorescence quenching process belonged to static quenching. The value of 1 for the binding site and positive relation of K_a to temperature demonstrating PL might combine one DMY to produce a stable complex, which was further evidenced by K_q values exceeding 2.0×10¹⁰ L·mol^-1·s^-1. According to the Van't Hoff equation, the results of thermodynamic parameters ΔS=0.201 4 J·mol^-1·K^-1, ΔH=32.311 kJ·mol^-1 and ΔG<0 under 293 and 310 K elaborated that the binding force was mainly hydrophobic force and a spontaneous and exothermic process. The binding distance r=1.475 nm reflected the possible non-radiative energy transfer from PL to DMY based on the theory of Förster's non-radiative energy transfer. Both synchronous fluorescence spectroscopy and UV spectroscopy results ascertained that the amino acid residue microenvironment and secondary structure of PL changed after the interaction with DMY. The former displayed DMY could bind to the surroundings of tryptophan (Trp) residue in PL by 2 nm red-shifts of the spectrum (Δλ=60 nm), while the latter uncovered the π→π^* transition of PL interacted with DMY. 3D fluorescence spectroscopy found the polarity of PL increased after hydrophobic interaction with DMY by 10 nm red-shifts of peak1 causing 51.38% decrease of fluorescence intensity and 5 nm bathochromic shifts of peak2 with 41.93% loss of fluorescence intensity. Moreover, the molecular docking results showed that the DMY binding site was located in the pocket of PL, which was formed by PHE77, PHE215, TYR114, ILE209, and PRO180 amino acids. The hydrogens in amino acids of HIS263, TYR114, SER152, and PHE215 could be linked to —OH of DMY on A ring C5, B ring C4′, C ring C3 and C ring —C＝O via hydrogen bonds, and other amino acids including THR78, LRU213, GLU179 and ALA178 may formed van der Waals forces with DMY. The experimental data obtained to a deeper understanding of the lipid-lowering molecular mechanism of DMY and its unique structure provides a theoretical basis for drug synthesis and screening of natural inhibitors.