A new method using gel permeation chromatography (GPC) cleanup followed by High performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) has been established for quantitative determination of 50 kinds of Pesticide residues in tobacco. In the method, the samples were extracted ultrasonically with ethyl acetate-cyclohexane (1∶1, v/v), 5 mL extract was injected into a 400mm?5mm S-X3 GPC column, with ethyl acetate-cyclohexane (1∶1, v/v) as the mobile phase at a flow rate of 5 mL/min. The 12–35 min fraction was collected for subsequent analysis. The portions collected from GPC were dissolved with methanol after taking the extract to dryness with nitrogen gas. The correlation coefficient of each pesticide was r2>0.99. The relative standard derivations(RSD) of Pesticide residues were between 1.00 % and 10.80 % , the recoveries fell within 86.2 %～108.4 %, the limits of detection for the method were 0.01～10.00 礸/kg..