A method basing on high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for determining the antiseptic residues in tobacco. The analytes extracted from tobacco samples were separated on C18 column (150 mm×2.1 mm×3 μm) using acetonitrile-water (containing 0.1 % formic acid) as mobile phase. The antiseptics were determined by a tandem mass spectrometer with internal standard under multiple reaction monitoring (MRM) mode. The 145 domestic and imported tobacco samples were determined with this method. The results showed that: 1) The linear range was 5-200 ng/mL (R²>0.999). The average recoveries ranged from 85.3 % to 118.8 % with the relative standard deviations (RSD) less than 7.7 %. 2) No antiseptic residue was detected in 52 tobacco samples and 30 cigarette samples, which accounted for about 52 % of all leave samples and 66.7 % of all cigarette samples; all the mentioned antiseptics, except benomyl, were detectable in samples. This method was suitable for the fast determination of the residues of the 6 antiseptics in tobacco and tobacco products.