Abstract: In order to improve the quality of germinated tobacco seeds and enhance the quality of seedlings, the Zhongyan100 and Zhongchuan208 were used as experimental materials to investigate the effects of gibberellin (GA3) with the different concentrations and the biocontrol agent "Haiyinong" treatment on seed germination potential, germination rate, germination index, seedling root length, maximum leaf area, seedling root dry (fresh) weight, dry (fresh) weight of above-ground part, antioxidant enzyme activities and malondialdehyde content. The results showed that the optimal concentrations for Zhongyan100 and Zhongchuan208 were GA3 100 mg/L and 50 mg/L, Haiyinong 2 g/L and 1 g/L, respectively. Most treatments of GA3 and Haiyinong significantly promoted the growth of seedlings of both two tobacco varieties. The seed germination index and seedling growth index were evaluated by membership function method. From the perspective of promoting seed germination and breeding strong seedlings, the best treatment for Zhongyan100 was GA3 200 mg/L, followed by Haiyinong 1g/L. The best treatment for promoting Zhongchuan208 was Haiyinong 1g/L, followed by Haiyinong 20g/L. The results of comprehensive evaluation of MDA and antioxidant enzyme activities in seedling leaves under low temperature stress showed that Haiyinong 2 g/L was the best treatment to improve the cold tolerance of Zhongyan100, followed by 100 mg/L of GA3. The best treatment to improve the cold resistance of Zhongchuan208 was GA3 100 mg/L, followed by Haiyinong 8 g/L. In summary,the types and concentrations of the initiator, which had the best effects on seed germination, seedling strengthening and anti-chilling effect of different tobacco varieties, are different, and the use of initiator should be flexibly selected according to the actual situation in the actual production.