Combined with the modified single-chamber smoking machine, the cigarette mainstream smoke puff-by-puff detecting system is established by utilizing the overtone absorption characteristics of CO and CO2 near 1579 nm based on tunable diode laser absorption spectroscopy (TDLAS) technique, wavelength modulation technique and second harmonic detection method. Experimental results show that the linearity between gas concentration and their second harmonic intensity is better. The system response speed is quick, and the precision can reach the detection requirements. The stability, repeatability and sensitivity of puff-by-puff detecting are all improved. The release amount of CO and CO2 in mainstream smoke for the same type of cigarettes increases with puff number except for the first puff. Both the puff-by-puff release amount and increasing rate of CO2 are larger than that of CO, furthermore, different types of cigarettes follow the same rules as well. The established system has the advantages of high detection speed, high time resolution, simple structure, and so on. It has a good application rospect in the field of rapid detection of harmful substances in cigarette smoke.