Abstract: Four nitrogen levels of N 0, 45, 90 and 135 kg/ha were setup to investigate the free amino acid and protein contents in Honghuadajinyuan (Hongda) tobacco at different nitrogen application levels. The results showed that histidine and asparagines content of tobacco increased significantly with the nitrogen application level. Glutamine and arginine contents in the 90 kg/ha N treatments were the highest, and decreased in the 135 kg/ha N treatment. Lysine content was decreased with the nitrogen levels increased. The total amount of free amino acids and protein content of tobacco increased significantly with the nitrogen application level, with up to 9.617 mg/g and 10.56% in the 135 kg/ha N treatment. The quality of aroma and sensory quality reached the highest in 90 kg/ha N and the lowest in 0 N treatment. Under the conditions of this experiment, the total amount of free amino acids and protein content of tobacco increased significantly with the nitrogen application level, but the contents of free amino acids in different species showed different expression with nitrogen application level, the sensory quality of tobacco was the best in 90 kg/ha N.