Abstract: In order to quantify tobacco terpenoids, a method for simultaneous determination of 21 terpenoids, including t, t-farnesol, α-phytol, cis-abienol, β-phytol, (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (α-CBD), (1S, 2E, 4R, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (β-CBD), sclareol, labd-13-ene-8,15-diol, squalene, 2,3-oxosqualene, dihydrotachysterol, cholesterol, brassicasterol, ergosterol, campesterol, stigmasterol, lanosterol, β-sitosterol, β-amyrin, α-amyrin and chenodeoxycholic acid in tobacco was developed using gas chromatography triple quadruples mass spectrometry (GC-MS/MS). The characteristic fragment ion peaks of the 21 terpenoids were obtained under selective reaction monitoring (SRM) mode in positive channels. The results noted that under the optimized conditions, the determination coefficients (R²) of the 21 terpenoids were 0.999 0-0.999 8 in the linear ranges. The limits of detection (LOD) for analytes ranged from 12.7 to 308.7 ng·g^-1, and the limits of quantification (LOQ) were 35.8-984.6 ng·g^-1. The intra- and inter-day relative standard deviations (RSDs) were 0.19%-8.28% and 1.02%-9.35% respectively. Mature fresh tobacco leaves from Yunnan, Henan and Guizhou Provinces were analyzed using this method. The results showed that the total amount of α-CBD was Yunnan (98.83 μg·g^-1) > Henan (57.04 μg·g^-1) > Guizhou (38.61 μg·g^-1), and there was significant difference among them. In particular, the terpenoid contents in leaves from Guizhou differed substantially from Yunnan and Henan, and that in leaves of cv. Nanjiang No.3 from Zunyi was significantly different from the other three varieties. This method uses only a simple pretreatment and is reproducible, can cover a wide range of terpenoids for batch analysis of tobacco leaves.