A method for the simultaneous determination of 34 pesticides in urine by QuEChERS coupled with on line gel permeation chromatography-gas chromatography-mass spectrometry (GPC-GC/MS) was developed.Methods The effect of extraction and purification which the extraction and combinations of sorbent involved were investigated. The identification and quantification were performed by GPC-GC/MS in SIM mode and external standard method. Results The LOD of this method ranged from 0.05-3.2 μg/kg (S/N=3) in urine, the average recoveries of 34 pesticides for samples spiked at 0.05 μg/g ranged from 81.1%-108.8% with the relative standard deviations (RSD,n=6) less than 10.4%.Conclusion The convenient operation and versatility of this method is suitable for the fast screening and detection of 34 pesticide residues in urine.