Abstract: Glandular trichome exudates and nicotine play a significant role in enhancing the aroma quality and stress resistance of flue-cured tobacco. To achieve a coordinated improvement, this study used the tobacco variety K326 as material. Through EMS mutagenesis technology, loss-of-function mutants for NtCycB2 gene (negative regulator of glandular trichome development) and NtJAZ1 gene (inhibitor of nicotine synthesis) were obtained. By continuous backcrossing combined with molecular marker-assisted selection, an improved line, named NK3, with high glandular trichome density and elevated nicotine content was successfully developed. Field trials indicated that the agronomic traits of NK3 were comparable to those of the control variety K326, but its glandular trichome density increased by 43.3%, total glandular trichome secretion amount increased by 27.5%, and the content of solanone (a degradation product of cembranoids) in cured leaves increased by 27.4%. The nicotine content in fresh and cured leaves of NK3 increased by 41.1% and 39.81%, respectively, compared to the control. Furthermore, sensory evaluation quality was superior to that of the control. Transcriptomic and metabolomic analyses showed significant enrichment of terpenoids, flavonoids, and other secondary metabolites in NK3 leaves. In summary, the new line NK3 has achieved a coordinated improvement in glandular trichome density, glandular trichome secretions, and nicotine content while maintaining stable agronomic traits. This not only provides new materials for the quality improvement of flue-cured tobacco but also offers a theoretical basis for elucidating the molecular mechanisms regulating glandular trichome secretions and nicotine content.