Abstract: Tobacco mosaic virus (TMV) is one of the main diseases of tobacco. The study aims to investigate the anti-TMV activity of chemical compound from tobacco by using Bioassay-guided Method and provide the basis for anti-viral biological pesticides from tobacco. The constituent of anti-TMV activity was isolated from Nicotiana glutinosa in Yuxi by bioassay. A kind of alkaloids that include three new compounds (1-3)(S)-Nkolbisine-β-D-glucoside, (R)-Nkolbisine-β-D-glucoside, Lepabisine A and nine known ones (4-12), were isolated from the whole-plant of Nicotiana glutinosa by various chromatographies such as silica gel, Sephadex LH-20, RP-18 column chromatography and HPLC. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. These compounds were also evaluated for their anti-tobacco mosaic virus (anti-TMV) activity. The results revealed that compounds 1 exhibited high anti-TMV activity with inhibition rate of 93.1%, 81.6% and 71.5%, respectively. The rates are higher than those of positive control. The other compounds also showed potential anti-TMV activity. This is the first report on the defense chemicals in tobacco, and provides scientific basis for the next systemic research.