Abstract: Effects of nitrogen application rate on physiological characteristics of flue-cured tobacco leaves at the maturing stage were studied by surveying chlorophyll content, nitrate reductase(NR) activity, soluble protein content and free amino acid content of fresh leaves of Zhongyan 100, a variety planted in Xuchang city of Henan Province, from different positions and in different periods every 5 days, form April to September, 2012. The results showed that during the gradually mature process of tobacco, effects of nitrogen application rate on soluble protein content and chlorophyll content of the upper tobacco leaves were not significant, but it was significant on NR activity and free amino acids content. The effect of nitrogen application rate on soluble protein content of the middle tobacco leaves were not significant, but it was significant on NR activity, chlorophyll content and free amino acids content. Only appropriate amount of nitrogen application could produce high quality tobacco leaves. The treatment of 45 kg/hm² pure nitrogen was the best.