The flue-cured tobacco variety K326 was used to study the changes in the accumulation of potassium in interlayer of leaves at the mature stage of tobacco under different application dosage of nitrogen and its correlation with parameters of canopy spectrum to real-time and nondestructively monitor the accumulation of potassium in flue-cured tobacco in field. The results showed that the accumulation of potassium in the upper layer of tobacco plants was not correlated with spectral reflectance in most bands except for a few bands. The accumulation of potassium in the middle and lower layers of tobacco plants was significantly negatively correlated with the spectral reflectance in the visible light band， and significantly positively correlated with the near-infrared band. The optimal spectral parameters for estimating the accumulation of potassium in different layers of leaf were not completely consistent. The optimal spectral parameter for estimating the accumulation of potassium in the upper， middle， and upper middle layers of tobacco plants was the ratio vegetation index RVI（810，680）， while the accumulation of potassium in the lower， middle lower， and upper middle lower layers of tobacco plants was most correlated with the red edge amplitude （D_λRed）. The established model for estimating the accumulation of potassium in the middle， lower， upper， middle， and lower layers of tobacco leaves was y=-0.002x²+0.159x-0.6531 with R² of 0.903^**， y=8.8736x²-4.1032x+0.5164 with R² of 0.618^**， y=0.0856x^1.29 with R² of 0.744^**， y=6.6558x²+0.5056x-0.3657 with R² of 0.821^**， y=0.2803x^0.5774 with R² of 0.938^**， and the model was tested to be stable. It is indicated that the established model for estimating the accumulation of potassium with parameters of spectrum can effectively invert the accumulation of potassium in the middle and lower layers and the whole plant of flue-cured tobacco.