Abstract: To further increase the mass fraction of glandular trichome secretion of Y2001, a mutant of NtCycB2 gene, which regulates glandular trichome formation, was created by EMS mutagenesis. The mutant was serially backcrossed with Y2001 as the recurrent parent, resulting in the modified line HY2001 with multi-glandular trichome orientation. The observation indicated that the total density of glandular trichomes of HY2001 increased by 117% compared with Zhongyan 100, in which the density of both long stalk and short stalk glandular trichomes increased significantly, while the density of non-glandular trichome type decreased significantly. The total density of HY2001 glandular trichomes increased by 77% compared with Y2001, in which the density of long stalk and short stalk glandular trichomes increased significantly, and the density of non-glandular trichomes increased slightly. The results of the chemical components on the leaf surface showed that the secreted components of HY2001 were the same as those of Y2001 and Zhongyan 100; however, its secretion content was significantly higher, especially the cembranoid diterpene content was 3.03 and 1.98 times as that of Zhongyan 100 and Y2001, and the sucrose ester content was 3.43 and 2.63 times as that of Zhongyan 100 and Y2001, respectively. The expression levels of diterpenoid biosynthesis related genes NtCBTS, NtCYP71D16, NtABS and sugar ester biosynthesis related genes NtASAT1 and NtASAT2 were upregulated in HY2001. The results of the drought stress experiment suggested that compared with Y2001, the wilting degree of HY2001 leaves was less and the antioxidant capacity of HY2001 line was significantly higher under drought stress. After inoculation with Phytophthora parasitica, the infection level of the stalk base of HY2001 plant was lower and the expression levels of resistance related genes NtPR1b and Nthsr203J were higher as compared to Y2001. The results of the aphid inoculation experiment showed that HY2001 was less attractive to aphids and the aphid reproduction rate on HY2001 was lower as compared to Y2001. The density of glandular trichomes and the secretion content of HY2001 increased significantly, and its resistance to drought, aphid and black shank improved. In conclusion, HY2001 has a great potential in tobacco growing areas in the central of Henan Province.