The UPLC-QE-Orbitrap-MS assay was used to compare the 2,5-dimethoxyfurano［4″,5″:3,4］chalcone (1) samples cultured in rat,mouse,rhesus monkey,Beagle dog and human liver microsomes for 0 min and 60 min in vitro,and compare the plasma,feces,and urine samples of intravenous injection of the compound and blank C57 mice to study its metabolites in vitro and in vivo.It was preliminarily concluded that there were two kinds of metabolites M1 and M2 in vitro after incubation,and five kinds of metabolites M1,M2,M3,M4 and M5 in vivo.M5 was further metabolized from M1,and the metabolite M1 was obtained by organic synthesis. Ultrafast liquid chromatography-mass spectrometry (UFLC-MS/MS) detection method was used to determine the plasma concentrations of 1 and M1.The change trend of the compound and M1 in rats was quantitatively studied and the pharmacokinetic parameters were calculated.Rats were randomly divided into groups and given drugs,and blood was collected at a set series of time points.The plasma concentrations of 1 and M1 were determined.The pharmacokinetic parameters were calculated by DAS2.0 software.The C_max of 1 was 405.96 μg/L,T_max=0.083 h,AUC_0-t=190.64 μg/（L·h）,T_1/2=3.74 h.The C_max of M1 was 281.291 μg/L,T_max=0.083 h,AUC_0-t=561.30 μg/（L·h）,T_1/2 =3.01 h.These results showed that 2,5-dimethoxyfuran［4″,5″:3,4］chalcone undergoes reduction,demethylation,ring opening,and hydroxylation reactions during the metabolism of the body.The main metabolite is M1,and M1 is further metabolized to M5.This study laid a material foundation for revealing its pharmacological effects.