With a combination of QuEChERS method and gas chromatography-tandem mass spectrometry (GC-MS/MS) technique, three sample treatment methods (solvent exchange method, toluene dilution method, hexane liquid-liquid extraction method) were established to analyse more than one hundred pesticide residues in tobacco. The 155 pesticide analytes contained 58 organophosphorus, 28 organochlorine, 10 pyrethroids, 10 acyl amide, 11 carbamate, 7 dinitroaniline and 31 other family pesticides. Comparison of the three treatment methods was investigated on performances including matrix effect, co-extracted matrix, peak interference, recovery and the limit of quantification (LOQ). The hexane liquid-liquid extraction method gave the cleanest extract but poorer recovery; the solvent exchange and toluene dilution method could guarantee recoveries in the range of 70% and 120% for most of the 155 pesticides. We found that organophosphorus, acyl amide and carbamate pesticides exhibited relative strong matrix effects compared with organochlorine and pyrethroid pesticides. Toluene dilution method was recommended to analyse organophosphorus pesticides, and hexane liquid-liquid extraction (LLE) method was recommended to analyse organochlorine and pyrethroid pesticides.