Flue-cured tobacco cultivar Xiangyan7 was used as the test material. Four treatments were set up: T1, treated at 4 ℃ for 4 d, followed by continuously spraying of 0.01 mg/L EBR for 4 d; T2, treated at 4 ℃ for 4 d, followed by continuously spraying of 340 mg/L H2O2 for 4 d; CK1, treated with low temperature(4 ℃) for 4 d, followed by continuously spraying distilled water for 4 d; CK2, continuously spraying distilled water for 4 days without low temperature treatment. Biomass, antioxidant enzyme(SOD, POD, CAT) activity, MDA content, osmotic regulatory substances and root activity of tobacco seedlings were measured. The results showed that compared to CK2(normal growth control), root activity and biomass of Xiangyan7 seedlings under 4 ℃ low temperature stress were decreased; while CAT activity, MDA content, soluble sugar and proline content were increased and SOD activity, POD activity and soluble protein content had no significant change. Compared to CK1(low temperature treatment), the root activity, root shoot ratio and protective enzyme activity of tobacco seedlings sprayed with EBR and H2O2 after low temperature stress was relieved, were significantly increased; and dry matter quality in T1(with EBR) and T2(with H2O2) increased by 40% and 20% respectively; soluble sugar in T1(with EBR) and T2(with H2O2) increased by 66.3% and 51.3%, respectively; MDA content in T1(with EBR) and T2(with H2O2) decreased by 37.8% and 37.5%, respectively. On the 8th and 12th day after low temperature treatment, SOD, POD, soluble sugar and proline showed no significant difference while soluble protein decreased significantly in EBR and H2O2 treatment, compared with normal growth treatment(CK2). Conclusion: after the normal growth temperature was restored, spraying exogenous EBR and H2O2 could accelerate the recovery of the physiological indexes of tobacco seedlings to a certain extent, and EBR showed better effect.