Abstract: The study aims to investigate the relationship between water stress and biomass accumulation and physiological metabolism of flue-cured tobacco in different growth stages. Using the flue-cured tobacco ‘Yueyan 98’ as the material and the method of greenhouse potted, the changes of dry matter accumulation and the key enzyme activities of carbon nitrogen metabolism of tobacco plant were studied under the condition of water stress in the stage of root spreading, fast growing and tobacco leaf maturity. The results showed that the inhibition effect of water stress in the late stage of root spreading on the dry matter accumulation in field tobacco plant was lagging and irreversible; moderate water stress in the stage of fast growing could significantly inhibit the accumulation of dry matter in the stem of the tobacco plant, but could be reversed, and the chlorophyll content and nitrate reductase activity in the leaves were relatively high, while the amylase activity was in a low level when the leaves of tobacco plant entered the mature stage; moderate water stress in the stage of tobacco leaf maturity significantly reduced dry matter accumulation in root and stem organ and the whole plant, but had no obvious effect on dry matter accumulation in leaf organ. In production practice, the water management in the stage of the root spreading and tobacco leaf maturity should be attached with great importance to ensure that the tobacco plant is not affected by drought, and that moderate drought stress in the stage of fast growing can improve the maturity of tobacco leaves in mature stage.