OBJECTIVE To develop a high performance liquid chromatography (LC) method for simultaneously determination of polycyclic aromatic hydrocarbons (PAHs) in serum. METHODS PAHs could be separated completely by using C18 column and detected by fluorescence detector at wavelengths (Ex = 340 nm, Em = 425 nm). The mobile phase A was acetonitrile and mobile phase B was water with a flow rate at 1.0 ml/min. RESULTS The concentration of PAHs achieved a good linear relation with the peak area. The correlation coefficient was in the range of 0.9941-0.9999. The recoveries were in the range of 91.4%-99.8% and the RSD were in the range of 2.56%-12.7%. The detection limits were in the range of 0.05-0.10 ng/ml. CONCLUSIONS The method was simple, rapid, sensitive and accurate, and suitable for the detection of PAHs in serum.