Objective to study the enzyme kinetics of paeonol in homogenized liver of rats, and to evaluate the effect of the CYP450 enzyme on the metabolism of paeonol. The metabolites of paeonol were also identified. Methods hplc method was established for quantitative analysis of remaining concentration of paeonol in liver homogenate, and enzyme kinetic parameters were calculated. The metabolites of paeonol in liver homogenate were identified with HPLC/MS/MS. Results The metabolism of paeonol was significantly promoted by the enzymes subtypes of CYP450(CYP4503A) which was induced by dexamethasone, and there was no change in the metabolism of paeonol in liver homogenate pretreated by the enzymes subtypes of CYP450 (CYP4502B) which was induced by phenobarbital. The metabolites of paeonol in liver homogenate were 4- methoxyacetophenone-2-O-glucuronide and 2,4-dihydroxyacetophenone-5-O-sulphate. Conclusion CYP3A may be mainly involved in the metabolism of paeonol, and then to provide scientific evidence for rational administration and metabolic mechanism.