This study was conducted to evaluate the chemiluminescence nitrogen oxide analyzer model 200E for nitrogen oxide analysis in mainstream tobacco smoke. Nitric oxide(NO) analysis in tobacco smoke depend on the type of NO analyzer and interface materials. For this study, 20 channel rotary type smoking machine(Borgbaldt RM20 CS) was used. Three kinds of interface materials, such as gas washing bottle, tedler gas collection bag and teflon vessel were tested in this study. Also, the effects of cigarette number, tar level of cigarette, and stirring device on the analysis of NO were tested. To use chemiluminescence NO analyzer for analysis of NO concentration in mainstream smoke, teflon vessel was the most stable and convenient interface material in case of different flow rate between smoke generation by smoking machine and air sampling by NO analyzer. Though the cigarette number of smoking affected on the analysis of NO concentration in mainstream smoke, the tar level of cigarette did not affect on the analysis of NO concentration in tobacco smoke. The effect of stirring device during smoking was not significant change to analyze of NO. Comparing to the results of analysis of reference cigarette, Ky2R4F, this method was adaptable to all of cigarette to analyze NO concentration in mainstream tobacco smoke. This result indicated that using teflon vessel for interface, 20 cigarettes for one smoking, and stirring condition was the best environment to analyze NO concentration in mainstream tobacco smoke by using chemiluninescence nitrogen oxide analyzer model 200E.