Aqueous two-phase solid-phase extraction (SPE)-TFA trifluoroacetylation (TFA)-GC/MS was developed for determination of glycosides in tobacco. The sample was extracted by microwave two-phase extraction, and separated by 0.45 μm membrane, and then by HP-20 SPE column, which was eluted by N-pentane dichloromethane and methanol, the eluent was collected, concentrated and dried to obtain the glycoside extract. The acetylation of trifluoroacetamide (MBTFA) was used as a derivatization method, the quality of the derivative was compared with the certified reference material of glucoside and the NIST spectrum library, and the area ratio of the derivative to the internal standard of 2-nitrobenzene-β-D-glucoside was quantitatively analyzed, by which 18 glycosides in tobacco were identified. The method was applied to the determination of flue-cured tobacco(K326), burley tobacco(Kenek) and oriental tobacco (Basma), and 18 glycoside flavor components were identified. The glycoside types include 14 glucosides, 1 rutin, and 3 nesteosides; and the aglycone types include aliphatic alcohols, aromaticity, norisoprenoids, sesquiterpenoids, and polyphenols. The content of glycosides in flue-cured tobacco was the highest, and in oriental tobacco the lowest.