Abstract: In order to investigate the puff-by-puff release of six chemical components(NNK, BaP, crotonaldehyde, phenol, ammonia and HCN) in mainstream cigarette smoke, a smoking machine RM20 H was modified to automatically trap mainstream cigarette smoke on a puff-by-puff basis. By referring to the relative national and industrial standards, the mainstream smoke of four commercially available domestic cigarettes of different specifications were tested, the said components in mainstream cigarette smoke were collected puff-by-puff and determined, and their variation was analyzed. The results showed that: 1) With the proceeding of puffing, the crotonaldehyde release per puff remained constant, while the other components increased in different ways: NNK increased linearly, BaP did logarithmically or exponentially, phenol did polynomially(n ≥ 5), ammonia did logarithmically, and HCN did exponentially. 2) A relatively higher correlation existed between the puff-by-puff release of the said components and sequential number of puffing, the multiple correlation coefficient(R) was in the range of-0.486(HCN) < R < 0.995(NNK). 3) This method realized the automatic puff-by-puff trapping and quantitative analysis of mainstream cigarette smoke, it was suitable for analyzing the said chemical components in mainstream smoke on a puff-by-puff basis.