Abstract: In order to increase the density of glandular trichomes and nicotine content of flue-cured tobacco to improve plant resistance and tobacco quality, hairy mutant line K326 with NtCycB2 knockout was used as the study material, and NtJAZ1 gene was edited by CRISPR/Cas9 technology. NtCycB2 and NtJAZ1 gene co-knockout line J-21 was selected and its agronomic traits, glandular trichome development, nicotine biosynthesis and leaf chemical components were studied with K326 and hairy K326 as controls. The results showed that the growth and development of J-21 plants were normal, and its main agronomic characteristics were not significantly different from those of K326 and hairy K326. The density of leaf glandular trichomes of J-21 was lower than that of hairy K326, but significantly higher than that of K326, and the content (mass fraction) of glandular trichome secretion was increased by 156.35%. At the bud stage, the nicotine content of J-21 was significantly higher than that of K326 and hairy K326, but the rate of increase decreased after topping. The nicotine content in the mature stage was 37.14% and 32.28% higher than those of K326 and hairy K326, respectively. The chemical components in cured leaves were harmonious, and the nicotine content of J21 was increased by 39.81% and 45.32%, the content of cembranoid degradation product solanone was increased by 217.49% and 48.45% compared with K326 and hairy K326, respectively. These results indicated that the co-knockout of NtCycB2 and NtJAZ1 can effectively increase the glandular trichome density of tobacco leaves, promote the accumulation of glandular trichome secretion content and nicotine without affecting the normal growth and development of tobacco plants, which is one of the effective methods to simultaneously improve tobacco resistance and characteristics.