The experiment was set up for examining the physiological and biological indexes quickly and exactly, for obtaining information of tobacco-field fertilizing and tobacco growing. The ASD Field spec FR 2500 was used to measure spectra reflectance of flue-cured tobacco and the relationship between hyperspectral parameters and biochemical contents (total nitrogen, chlorophyll, carotenoid), and physiological indexes (fresh weight, dry weight, moisture content) of flue-cured tobacco leaves was studied by correlation and stepwise regression statistic methods at different nitrogen and potassium levels. The results indicated that the spectra curves of different treatments had obvious rules and great diversities. There were high correlations between different types of spectra parameters and ten physiological and biochemical indexes of flue-cured tobacco leaves. Hyperspectral characteristic variables of ten physiological and biochemical indexes were found through stepwise regression, and SDr/SDb was the characteristic variable closest to seven biochemical contents. Simultaneously, the R2 and regression coefficient of equations reached 0.05 significant level and the equations had good estimating effects through the examination of other samples. Accordingly, this study suggested that the ten physiological and biochemical indexes could be estimated quickly by the estimating models, at the same time nitrogen-potassium fertilization and growth condition of flue-cured tobacco could be inspected.