Abstract: 8306 Line is a flue-cured tobacco breeding material with the flavors of air-cured and sun-cured characters. In previous studies, an NtCPS2 gene mutant line, named as 8306-1, was created to inhibit the synthesis of labdane diterpenoids, the representative flavor substances of air-cured and sun-cured tobacco. To further increase the mass fraction of cembranoid diterpene and sucrose ester in 8306-1 line, gene editing technology was used to knock out the NtCycB2 gene of 8306-1 line, and a homozygous mutant strain 8306-2 was obtained. Compared to 8306 and 8306-1, the growth and development of 8306-2 line was normal, and there were no significant differences in agronomic traits. The density of long stalk secretory glandular trichomes of 8306-2 line was significantly higher than those of 8306 and 8306-1 lines, and 8306-2 line had more branched glandular trichomes. The compositions of glandular trichome secretions of 8306-2 and 8306-1 lines were the same, which both lacked the cis-abienol and labdenediol in 8306. Meanwhile, the mass fraction of leaf surface chemistry of 8306-2 line were higher than those of 8306 and 8306-1 lines, among which the content of cembranoid diterpene increased by 95.7% and 93.4% respectively, and the content of sucrose ester increased by 60.8% and 62.5% respectively. qRT-PCR analysis found that the expression levels of the four key diterpene synthesis enzyme genes NtCBT, NtCYP71D16, NtCPS2 and NtABS of 8306-2 line were significantly higher than those of 8306-1 and 8306 lines, while there was no significant difference in the expressions of the upstream genes NtGPPS and NtGGPPS for diterpene synthesis. It indicated that the co-knockout of NtCycB2 gene and NtCPS2 gene of 8306 line significantly increased the density and secretion of tobacco glandular trichomes on the basis of effectively inhibiting the synthesis of labdane.