Abstract: In order to accurately analyze the puff-by-puff release characteristics of CO in gas phase of mainstream cigarette smoke,an on-line puff-by-puff analysis system for cigarette smoke was established,which incorporated tunable diode laser absorption spectroscopy(TDLAS) technology with a modified single port smoking machine.The effects of cigarette smoke remained in pipeline and filter pad holder on the results of puff-by-puff analysis were analyzed with the system.The puff-by-puff release and transfer tendency of CO in mainstream smoke of cigarettes of four different types were analyzed.The results showed that:1) The puff-by-puff transfer tendency of CO obviously differed before and after flushing the residual smoke from pipeline and filter pad holder.2) Under the flushing mode,the puff-by-puff release of CO in mainstream smoke decreased at the first and second puffs then increased gradually with the minimum at the second puff.Comparing with traditional analysis methods,the modified single port smoking machine combined with TDLAS technology can complete the analysis of CO in cigarette smoke quickly and avoid the interference from other components in mainstream cigarette smoke effectively.