Abstract: In order to realize the real-time and nondestructive monitoring of leaf area index (LAI) for flue-cured tobacco at mature stage, a field experiment was conducted to analyze spatial-temporal distribution of LAI at different leaf layers of tobacco plants with different nitrogen application rates. The results showed that the relationships of spectral reflectivity with LAIs of middle layer, middle-lower layers and top-middle-lower layers (canopy layers) of tobacco plants were similar, which were all significantly negatively correlated with spectral reflectivity in the visible light region, while significantly positively correlated within wave band of 760-1 300 nm. The similarity between the correlation curves of spectral reflectivity and LAIs of middle and canopy layers of the plants were high, and LAI of middle layer could reflect the LAI of the whole plant to a large extent. The correlations between RVI (810, 680) and LAIs of middle layer, top-middle layers and canopy layers of the tobacco plants were the highest, while the correlations between Dλ_Red and LAIs of lower layer and middle-lower layers were the highest. The quadratic curve model based on RVI (810, 680) could accurately predict the LAIs of middle layer, top-middle layers of the tobacco plants, and the power model based on RVI (810, 680) could efficiently inverse the LAI of canopy layers. The quadratic curve model based on Dλ_Red could efficiently inverse the LAIs of lower and middle-lower layers. Therefore, these spectral parameters could nondestructively monitor LAI of middle-lower layers of tobacco plants in real time.