Abstract: In order to qualitatively analyze tobacco aroma components accurately and rapidly, a gas chromatography-tandem mass spectrometry (GC-MS/MS) method was established based on the retention indices (RI) of 241 tobacco aroma components. The retention indices of 241 target components were determined first by using standard samples, which accurately differentiated isomers in those standard samples and to realize rapid adjustment of the acquisition time window of the multi-analyte GC-MS/MS analytical method. The results showed that: 1) Among the retention indices of 241 aroma components, the differences between experimental determined values and those from the NIST17 database were less than 10 for 212 of the components, and the retention indices of the other 25 components had not been recorded in NIST17 database. 2) Adding retention index successfully promoted the accuracy and reliability of qualitative analysis among isomers. 3) Retention indices of the targets were used to predict the retention times of the target compounds under different chromatographic conditions, the time deviations of 232 components were within 0.05 min, and that of the other 9 components were within 0.05-0.10 min. It proved that the retention index had good stability in complex tobacco matrix and could be used to accurately predict the acquisition time window in multi-analyte GC-MS/MS method rapidly.