Halogenated polycyclic aromatic hydrocarbons (HPAHs) have attracted extensive attention because of their high toxicity and bioaccumulation. However, there has been no report on the content of HPAHs in human tissues and the corresponding analytical method. In this study, a method for the simultaneous determination of 16 polycyclic aromatic hydrocarbons (PAHs) and 23 HPAHs in human serum was developed and validated. Simple and stable removal of interfering substances in complex serum and the detection of ultra-trace HPAHs are the key difficulties. After 0.5 mL serum was treated with formic acid and 10% isopropanol, samples were prepared by solid phase extraction (SPE) and analyzed by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). The recoveries of the method were 65-103%, with low detection limits of 0.001-0.019 ng mL-1. For HPAHs, the precision was in the range of 0.2-10% according to relative standard deviation (RSD). Subsequently, the developed method was validated for serum samples obtained in hospitals, and 8 PAHs and 12 HPAHs were detected. The concentration of ∑HPAHs was 23 ± 12 ng g-1 lipid in females and 21 ± 10 ng g-1 lipid in males, in which phenanthrene and anthracene halogenated derivatives were the main components. The level of HPAHs was correlated with PAHs, which was 23-119 times higher than that of HPAHs. The detected HPAHs contain highly toxic and persistent components, representing an ongoing human health risk, which should receive more attention.