Abstract: AIM　To develop a method for analysis of palmatine in serum by high performance liquid chromatography with fluorometric detector. METHODS　Separation was obtained by using a LiChrosorb SI 60 column(4.0 mm×200 mm, 5 μm). The mobile phase consisted of a mixture of dichloromethane-methanol-diethyl amine-acetic acid(90∶9∶0.4∶0.5) and the flow rate was 1 mL.min^-1. Excitation and emission wavelengths were set at 365 and 510 nm respectively. To 1.0 mL of serum containing palmatine was added 13-methylberberine (internal standard， IS), then extracted with 5.0 mL chloroform containing trichloroacetic acid. The organic phase was removed with nitrogen and the residue dissolved with 100 μL dichloromethane. After centrifugation, 20 μL of the lower layer was subjected to HPLC. RESULTS　The retention times of palmatine and IS were 8.4 and 7.1 min respectively. In serum the detection limit of palmatine was 0.1 ng.mL^-1. The extraction recoveries of palmatine and IS were over 85％. The relative standard deviations of within-day and between-day were 0.94％～1.85％ and 0.10％～6.47％ (n＝6) respectively. CONCLUSION This method is sensitive, simple and fast, so it can fit the need of palmatine pharmacokinetic research preferably.