Abstract: In order to quantify the pesticide residues in particulate matter of mainstream cigarette smoke, a procedure using acetonitrile-based extraction combined with dispersive liquid-liquid microextraction (DLLME) and gas chromatography-tandem mass spectrometry (GC-MS/MS) detection was developed for determining the residues of 63 pesticides. The effects of extraction solvent type and volume, extraction time were investigated and optimized. The total particulate matter of mainstream cigarette smoke collected by a Cambridge filter pad was extracted with acetonitrile under oscillation, the extract was further disposed by DLLME, the obtained tetrachloromethane extract was analyzed by GC-MS/MS and quantified by internal standard method. The results showed that good linearity was observed for all 63 analytes with the correlation coefficients ranged from 0.994 9 to 1.000 0. The limits of quantification were no more than 10 ng/cig, and the average recoveries were 75.6%-127.5% with RSD from 0.5% to 19.0% at three different spiked levels. This method is simple, accurate, sensitive and suitable for the determination of 63 pesticide residues in particulate matter of mainstream cigarette smoke.