In order to elucidate the influence of theanine on the growth and related physiological indexes of tobacco seedlings, Yunyan 87 was used to study the effects of different concentrations of theanine (0, 0.5, 1 and 5 mmol/L) on the growth characteristics and related physiological indexes of tobacco seedlings under the hydroponic cultivation condition. As results, theanine significantly decreased the root length, root dry and fresh weight, shoot dry and fresh weight, root-shoot ratio, and chlorophyll content; however, it significantly increased the content of soluble protein, proline, and GSH. The increase of SOD and POD activities and the decrease of CAT activity caused the unbalance of antioxidant enzyme systems, which ultimately led to the accumulation of H2O2 in tobacco seedlings, causeing lipid membrane damage. Furthermore, the increase of the relative conductivity and MDA content verified this conclusion. These results showed that application of exogenous theanine could make the antioxidant enzyme system imbalance and cause oxidation reactions. Therefore, we concluded that theanine might inhibit the growth of tobacco seedlings by disturbing the equilibrium of the antioxidant system.