Abstract: 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.