The quantitative relationship between vertical distribution of above-ground nitrogen accumulation and canopy reflectance spectra in flue-cured tobacco (Nicotiana tabacum L.) was studied. It aimed at determining the contribution of leaf layers especially middle layer and lower layer to canopy reflectance, and constructing vertical distribution inversion model of nitrogen accumulation in different leaf layers and its combination in flue-cured tobacco. The field experiments with different nitrogen levels were conducted. The time-course measurements were taken on canopy spectral reflectance and weights and nitrogen contents in different plant parts and leaf layers during the experimental period. By means of analysis of correlation with experimental results, it was found that RVI(810,680) could efficiently inverse the ANA in middle layer, upper-middle layer and the whole plant layers of flue-cured tobacco. DλRed could efficiently inverse the ANA of lower layer, middle-lower layer of flue-cured tobacco. It was concluded that ANA in middle, lower layer and the whole plantlets of flue-cured tobacco could be monitored directly by key vegetation indices.