Abstract: To investigate key compounds with spicy character in tobacco leaves, the sensory-oriented separation on robust flavor style flue-cured tobacco was conducted with gel permeation chromatography (GPC). The fractions with spicy character were located and assembled on the basis of taste assessment results, the obtained spicy characteristic component (SCC) was qualitatively analyzed by GC/MS. Four compounds were identified using SCC. According to the qualitative analysis, the sensory quality of standard samples of the four compounds was evaluated. The content of nicotine in SCC was determined, and SCC solution and recombined nicotine solution were prepared for taste dilution analysis (TDA) separately. The sensory effect of nicotine was further evaluated via blank cigarette flavoring. The result showed that:1) In the four compounds identified, only nicotine had the spicy characteristic. 2) Both SCC solution and recombined nicotine solution presented same spicy intensity and dilution factor in TDA, which suggested that nicotine was the main source of spicy feeling in SCC and the key compound with spicy character in flue-cured tobacco leaves. 3) Nicotine was closely related to the irritancy of cigarette smoke, which significantly increased the strength of cigarette smoke and its irritancy to oral, larynx and nasal cavity.