Accurate results are often hard to obtain in the determination of tobacco-specific N-nitrosamines (TSNAs) in mainstream smoke of Virginia type cigarette due to the low deliveries of TSNAs and the complicated matrix disturbance; therefore, a novel heart-cutting two dimensional liquid chromatography–tandem mass spectrometry (2DLC-MS/MS) method was developed. The method offers simple sample preparation involving only extraction and filtration, with almost no matrix interference and low quantitative limits compared with traditional methods. A strong cation exchange (SCX) column is utilised for first dimensional separation, which effectively removes the acidic and neutral components in smoke. To reserve TSNAs on the trap column, a compensate pump is applied for on-line dilution and pH adjustment during the period of TSNAs fraction transfer and enrichment. Next, a C18 column is employed for 2nd dimensional separation and coupled to tandem mass spectrometry under multiple reaction monitoring (MRM) mode. The 2DLC-MS/MS method with isotope deuterated internal standards is applied to the determination of TSNAs levels in mainstream cigarette smoke, the limits of detection of NNN, NAT, NAB and NNK were 0.023, 0.028, 0.019 and 0.028 ng/cig, and their spiked recoveries ranged from 93.6% to 108.6% with RSDs within 5.4%. The developed method is simple, automatic, sensitive, accurate and reliable; it is suitable for determining the deliveries of TSNAs in mainstream smoke of cigarette, especially Virginia type cigarettes.