Abstract: The changes of growth and physiological characteristics of tobacco seedlings in floating system were studied using cv. K326 as experimental material. The results indicated that when the seedling age was 70-80 days old, it had robust stem, heavier stems and root weight and higher ratio of root and shoot, and well developed root system, and its appearance reached the standard of strong seedling. Meantime, the seedling's physiological indexes, such as NR and INV activity, root vigor and active absorbing area were higher, SOD activity, ascorbic acids content, relative moisture content were relative high as well, it indicated that the seedling had good stress resistance; the praline content and electrolyte osmotic rate was relatively low, it indicated that the seedling was under little stress, and it was the proper time to be transplanted to fields.