Taking Yunyan99 as the material, the two-factor complete combination method was used to set up 3 soil moisture conditions including W1(soil relative water content 75%-85%), W2(soil relative water content 55%-65%), W3(soil relative water content 35%-45%); and 3 nitrogen application rates as N1(4 g), N2(5 g), N3(6 g), and a total of 9 treatments was set in pot experiments. After 15 days of vigorous growing stage of water control, catalase(CAT), peroxidase(POD), superoxide dismutase(SOD) activities and soluble protein, soluble sugar, proline(Pro), reduced glutathione(GSH), ascorbic acid(AsA), malondialdehyde(MDA), and superoxide anion radical(O2–) content were measured to explore the effect of water and nitrogen coupling on the osmotic regulators and stress resistance physiological characteristics of flue-cured tobacco. The results showed that under W3 conditions, nitrogen application could increase the soluble protein content by 42.86% and decrease the soluble sugar content by 12.32% compared with W3N1 treatment. Under W2 conditions, nitrogen application increased the activities of SOD and POD enzymes, which were 31.19% and 44.89% higher than those of W2N1 treatment, respectively. Under medium nitrogen conditions(N2), enzyme promotion effect exhibited, CAT activity in W1 or W2 treatment increased by 40.26% and 43.58% respectively, compared with W1N1 treatment. Compared with W1N1 treatment, the contents of Pro, GSH, MDA and O2- in tobacco leaves in W3 treatment were increased by 69.69%-222.49%, 15.2%-89.36%, 155.15%-162.37% and 24.89%-72.34%, respectively. The interaction analysis showed that the relative water content of soil had a greater effect on the activities of SOD and POD, and the contents of Pro, GSH, MDA, O2– and soluble sugar, while the nitrogen application rate had a greater effect on the content of soluble protein and CAT activity. The interaction effect of the relative water content and nitrogen application rate had a greater effect on the content of AsA. In general, the treatment with 6 g of nitrogen application had a better effect in promotion of antioxidant enzyme activity in flue-cured tobacco under mild water shortage, while the treatment with 5 g of nitrogen application had a better effect in increasing the content of osmotic regulators and antioxidant substances in flue-cured tobacco under severe water shortage.