To investigate the relationship between spectral characteristics of tobacco cultivars and their nitrogen use characters, four tobacco cultivars with different nitrogen use efficiency were used in a 15N pot experiment. The result showed that, in the visible light range, the spectral reflectance was lower in higher nitrogen level (N2) than N1, while opposite in near infrared range. The spectral reflectance of K326 and HD were lower than ZY100 and NC89 in visible light range, which closely related to their higher chlorophyll content. Both the nitrogen utilization rate of basal fertilizer and topdressing fertilizer in HD and K326 were higher than that in ZY100 and NC89 under N1 and N2 levels. The basal fertilizer use efficiency was negatively correlated with ρ550, and significantly positively correlated with RVI (800, 550), DVI (800, 550) and NDVI (800, 550) both in N1 and N2 level. The top dressed fertilizer use efficiency was significantly positively correlated with RVI (800, 550), DVI (800, 550) and NDVI (800, 550) in N1 level. Therefore, spectral characteristics can be an important method for diagnosing tobacco nitrogen metabolism characteristics.