Improving the nitrogen use efficiency in crops can significantly contribute toward reducing nutrient loss in soil and alleviating environmental pressure. It is well known that different morphologies and functional nitrogen play essential roles in the photosynthesis and respiration of crops. However, it is still unknown whether the differences in the accumulation and distribution of different morphological and functional nitrogen affect the nitrogen use efficiency in plants or not. In this study, we explored the impact of different application rates of pure nitrogen (0, 4, and 8 g/plant) on the accumulation of biomass, accumulation of nitrogen, and accumulation of different morphologies and functional nitrogen in different organs and nitrogen use efficiency of two flue-cured tobacco cultivars (Hongda and K326). Results showed that under CK (0 g/plant) and medium (4 g/plant) application rates of nitrogen fertilizer, the biomass and nitrogen accumulation in different organs and nitrogen use efficiency of Hongda was significantly higher than K326. Although under a high application rate of nitrogen (8 g/plant), the root system development of Hongda was reduced, but its leaf biomass and nitrogen accumulation were found higher than that of K326. Analysis of accumulation of different functional nitrogen in leaves showed that Hongda had a higher capacity to accumulate carboxylation system nitrogen, respiratory system nitrogen, and photosynthetic system nitrogen, which correlates with the higher photosynthetic and respiratory efficiency in Hongda. Further pairwise correlation analysis among nitrogen use efficiency, accumulation of different morphologies and functional nitrogen in different organs showed that nitrogen accumulation in the respiratory and carboxylation systems had a strong association with nitrogen use efficiency of flue-cured tobacco plants. This study revealed that Hongda could accumulate more carboxylation and respiratory system nitrogen which can promote stronger photosynthetic and respiratory abilities, resulting in higher nitrogen using efficiency of Hongda.