Abstract: Mainstream cigarette smoke (MCS) is very complex and highly dynamic aerosol. Some substances in smoke are highly reactive, resulting in a continuously changing mixture during smoke processing of trapping and separation. Because the conventional off-line methods can alter the smoke composition, an online method of extractive electrospray ionization mass spectrometry (EESI-MS) was established for analyzing of MCS. The MCS, generated by a single channel smoking machine, was directly introduced into EESI-MS without sample preparation, and the mass spectrum was obtained. MS was operated at collision induced dissociation (CID) mode for qualitative analysis of nicotine. This method was applied for the MCS monitoring on a puff-by-puff basis, and the results showed that the signal intensity of total ions (m/z 50-400) and ion of nicotine (m/z 163) increased with puffs, which indicated the trend of MCS emissions with the length of cigarette. Then, EESI-MS was used for analyzing of MCS of various cigarette brands. The result of principal component analysis (PCA) indicated that the EESI-MS method could be applied to differentiate MCS of various cigarette brands, the clustering analysis showed that the EESI-MS method could be applied to cluster the MCS of various cigarette brands and measure their similarity.