Abstract: Tobacco trichomes are closely related to disease resistance and leaf quality. To improve tobacco trichome density and create a high trichome density variety, CRISPR-Cas9 technique was used to knock out NtCycB2, which negatively regulated trichome formation, in cv. K326. A high trichome density line was obtained as a result, named as HK326 (Hairy K326). Furthermore, the effects of NtCycB2 on the agronomic traits and chemical composition of flue-cured tobacco were studied. The results showed that HK326 grew and developed normally, and its agronomic trait was no different to the controls. The trichome density on the surface of HK326 leaves increased. Glandular trichomes of HK326 had strongly biosynthetic and secretory functions, and cembranoids content significantly raised. The total content of neutral aroma components in flue-cured leaves of HK326 was increased, especially solanone and neophytadiene. Therefore, the trichome density, growing, development and main aroma component accumulation of K326 was promoted by knocking out NtCycB2, which suggested that HK326 had promising application potential in tobacco variety improvement.