Abstract: To improve the quality of primed tobacco seeds, especially the germination ability of old seeds, studies were conducted on the seeds of K326 and NC89 of different years. The seeds were primed by different concentration of MS medium, GA₃, IBA and 6-BA, and then the germination rate, physiological and biochemical indices and the expression of germination related gene were detected. The results showed that after priming treatment by proper concentration of each factor, the germination rate of flue-cured tobacco seeds were promoted, and the combination of different growth regulators (1/20 MS+100 mg/L GA₃+2 mg/L IBA+0.2 mg/L 6-BA and 1/20 MS+100 mg/L GA₃+2 mg/L IBA+2 mg/L 6-BA) had the best effects, After treatment of which the seeds geminated rapidly and each physiological and biochemical index was better than control significantly. What's more, the expression level of WRKY2 was enhanced remarkably after priming treatment.