Here, tobacco extract was produced by fermentation using Enterobacter, then the tobacco extract was prepared by concentration. Continuous flow was applied to analyze the chemical composition of tobacco extract; and the aroma components were also analyzed by GC-MS. The as-prepared tobacco extract was applied in the cigarettes, and its effects on sensory and smoke aroma components were also evaluated. Results showed that, after fermentation, the content of protein, starch, galacturonic acid, total plant alkali and amino acid were decreased by 28.95%, 19.44%, 18.91%, 3.71% and 37.45%, respectively. The tobacco extracts prepared by fermentation had a typical sweet flavor, and the characteristic flavor components of tobacco such as solanone, β-damascenone, and megastigmatrienone were significantly increased. The content of sweet fragrant substances such as farnesol, β-ionol, furfuryl alcohol, and furfural in cigarette smoke was increased, and the sensory quality was obviously improved.