[Objective] This study aimed to investigate the variation dynamics of total nitrogen and nicotine of burtey tobacco at various growing stages under different ni- trogen nutrition levels to provide reference for optimizing fertilization technologies for burley tobacco. [Method] Fresh burley tobacco leaves were collected at various growing stages and dried to measure the content of total nitrogen, protein and nico- tine by using INTEGRAL automated chemical analyzer and analyze the correlation with nitrogen application level. [Result] Regardless of the nitrogen application level, the content of total nitrogen and protein showed a downward trend since root ex- tending stage and reached the minimum at mature stage; the content of nicotine showed an upward trend since early vigorous growing stage and increased to the maximum at mature stage; the content of total nitrogen, protein and nicotine all in- creased after air curing; the content of total nitrogen, protein and nicotine showed positive correlation with nitrogen application level, while total nitrogen/nicotine was negatively correlated. The results indicate that nitrogen level is closely related to the total nitrogen and nicotine of burley tobacco at growing period and post-air curing, rational application of nitrogen is an important measure to regulate the content of total nitrogen and nicotine of tobacco. [Conclusion] This study provides scientific ba- sis for rational fertilization of burley tobacco.