Drought causes not only the decrease of tobacco yield and quality, but also the lowering of net photosynthetic rate, leading to reactive oxygen species accumulation and even the death of plants. Salicylic Acid is involved in regulating many plant physiological processes and has increasingly been applied to improve tolerance in plants exposed to drought stress. To explore the regulating mechanism of SA, flue-cured tobacco K326 was used in the hydroponic experiments to design PEG drought stress. The photosynthetic characteristics, antioxidant enzymes activities and osmotic regulatory substances contents of tobacco seedlings under drought stress were investigated after 0.3 mmol L -1 SA treatment. Transcriptome sequencing and GO/KEGG analysis were also performed. The main results showed that SA-applied greatly increased the activities of SOD, POD, CAT activity, Pn, proline and soluble protein by 44.27%, 50.18%, 26.23%, 45.74%, 34.67% and 24.91% while reduced the MDA content by 23.89%. GO and KEGG analysis showed that SA treatment was able to up-regulate the genes involved in photosynthesis, carbon metabolism, porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins. The conclusion is that SA application would effectively improve the ability of pigment biosynthesis and photosystem repair of tobacco under drought conditions, thus enhance the photosynthesis, reduce the accumulation of ROS and increase drought resistance, which would provide a measure for alleviating the damage of tobacco caused by drought stress.