Abstract: In order to analyze the pesticide residues in tobacco leaves, tobacco extract was directly analyzed by on-line gel permeation chromatography-gas chromatography-tandem mass spectrometry. This method was able to quantify the residue levels of 31 pesticides, and the optimal determination conditions were obtained via optimizing gel permeation chromatography collection time and multiple reaction monitoring parameters, which could influence the determination results. The results showed that: 1) Under the optimized conditions, the limits of detection of the method for the target analytes ranged from 1.95 to 212 ng/L, the determination coefficients (R²) were more than 0.99 for all the target analytes, the intra-and inter-day relative standard deviations were no more than 6.3% and 10.2%, respectively; and the recoveries of standard were from 82.2% to 121.5% for test samples. 2) The method was successfully applied to the analysis of pesticide residues in tobacco samples, the test results well agreed with those of existing standard methods.