High-speed counter-current chromatography (HSCCC) was investigated as a new sample pretreatment method for the analysis of trace polycyclic aromatic hydrocarbons (PAHs) in water environmental samples. The experiment was performed with a nonaqueous binary two-phase solvent system composed of heptane-acetonitrile. The HSCCC column was first entirely filled with the upper stationary phase of the and a large volume of water sample was pumped into the column while the CCC column was rotated at 1600 rpm. Finally, the trace amount of PAHs extracted and enriched in the stationary phase were eluted out by the lower mobile phase. and analyzed by gas chromatography-flame ionization detector (GC-FID) or gas chromatography-mass spectrometry (GC-MS) after concentration. The enrichment and cleanup of PAHs can be fulfilled online by this methodwithhigh recoveries (84.1%–103.2%) and good reproducibility (RSDs 4.9–12.2%) for 16 EPA PAHs under the optimized HSCCC pretreatment conditions. This method has been successfully applied to determine PAHs in lake waterwhere 8 PAHs were detected in the concentration of 40.9–89.9 ng/L. The present method is extremely suitable in the preparation of large volume of environmental water sample for the determination of a trace amount of organic pollutants including PAHs as studied in this paper.