Abstract: In order to investigate the puff-by-puff release characteristics of mainstream cigarette smoke, a photo-ionization-time-of-flight mass spectrometry(PI-TOF/MS) method was developed for on-line analyzing seven organic compounds(acetaldehyde, 1, 3-butadiene, acetone, isoprene, 2-butanone, benzene and toluene) in mainstream cigarette smoke. The constant volume sampling of mainstream cigarette smoke was achieved by a constant flow orifice, the smoke was directly introduced into the ion source of PI-TOF/MS through a sampling tube with a heating unit. The results showed that: 1) The intra-day and inter-day repeatabilities were good with the relative standard deviations(RSDs) less than 15%, exclusive of the first and the last puffs, where the deviations were larger due to cigarette lighting and variable puff volume, respectively. 2) The RSDs between the results of 1R5 F and 3R4 F reference cigarettes determined by this method and the mean test values of CORESTA collaborative experiments were less than 10%. 3) The deliveries of different compounds in single puff differed obviously, as did the deliveries between different puffs. This method is simple, fast, accurate, and suitable for the on-line puff-by-puff analysis of mainstream cigarette smoke.