An analytical method for the determination of benzo(a)pyrene and its metabolite 3-hydroxybenzo(a)pyrene in seafood was developed by high performance liquid chromatography with fluorescence detection (HPLC-FD).Methods The samples were extracted by 65% acetonitrile, treated by vortex blending and centrifugal precipitation. For benzo(a)pyrene and 3-hydroxybenzo(a)pyrene, the chromatographic separation were achieved in CenturySIL C₁₈-BDS(150mm×4.6mm, 5μm)column with methanol-water(pH 4.5)(85∶15),(87∶13)as mobile phase with a flow rate of 1.0ml/min, respectively. The benzo(a)pyrene and 3-hydroxybenzo(a)pyrene were detected under FD at λ_ex/λ_em=265nm/450nm, λ_ex/λ_em=365nm /450nm with an injection volume of 20μl, respectively.Results Benzo(a)pyrene was linear in range of 0.1～10.0ng/ml,r=0.9994and 3-hydroxybenzo(a)pyrene was linear in range of 1.07～100.7ng/ml,r=0.9921. For benzo(a)pyrene and 3-hydroxybenzo(a)pyrene, the limit of quantification was 0.1μg/kg, 0.2μg/kg, the recoveries were 96.8%～112.0%, 72.6%～83.3%, and the relative standard deviations was 6.0%, 5.2%(n=5), respectively.Conclusion The method is simple,rapid and highly sensitive. It is suitable for the determination of benzo(a)pyrene and 3-hydroxybenzo(a)pyrene in seafood.