Abstract: 【Objective】 In this study, we aim to report the production of transgenic tobacco ‘NC89' using Agrobacterium tumefaciens harboring the PBI121:CslA1 binary vector and the optimization of some transformation parameters to enhance transformation efficiency. 【Method】 By optimizing the Murashige and Skoog medium enriched with 6-BA(2.0 mg/L)and NAA(0.1 mg/L), we got a large number of seeding for subsequent usage and achieved the maximum number of shoots per explant. To find out the most effective condition for tobacco transformation, we examined some parameters, including the Agrobacterium concentration, pre-culture duration, Agrobacterium infection duration, co-culture period, treatment of selective media containing kanamycin and dark incubation in selective duration. Transgenic seedlings was obtained by controlling some transformation parameters including the Agrobacterium concentration, pre-culture duration, Agrobacterium transformation duration, co-culture period, to complete selection with media containing kanamycin. 【Result】 The highest transformation efficiency was obtained with two days of explant pre-culture, Agrobacterium concentration of D₆₀₀ being 0.7, Agrobacterium inoculation for 20 min. The appropriate concentration of kanamycin at the concentration of 100 mg/L was applied to screen transformed cells, and dark incubation for four days during co-cultivation increased the regeneration rate. Incubation for seven days in darkness after changing the selective medium shortened the callus induction duration. Putative transformants were verified by RT-PCR and Southern blot hybridization. 【Conclusion】 The present study achieved the overexpression of transgenes in tobacco and increased the transformation efficiency through optimizing conditions of the several key steps in gene transformation. This Agrobacterium-mediated transformation protocol could help a lot for the genetic application of A. tumefaciens mediated transformation in Nicotiana tabacum and related species.