Abstract: The near infrared (NIR) spectra from 534 domestic flue-cured tobacco samples of different crop years, habitats and grades were determined by rotating scanning with acousto-optic tunable filter-near infrared (AOTF-NIR) spectroscope and Unscrambler stoichiometry software. The calibration models for predicting total nicotine, total sugar, reducing sugar and total nitrogen in tobacco leaves were established by partial least square, and checked with 41 unknown flue-cured tobacco leaf samples. The results showed that the determination coefficients (R²) of the models for total nicotine, total sugar, reducing sugar and total nitrogen were 0.9869, 0.9851, 0.9733 and 0.9721, respectively; their average relative standard deviations were 4.21%, 2.23%, 2.91% and 3.60%, respectively; the method was suitable for rapid analysis of total nicotine, total sugar, reducing sugar and total nitrogen in tobacco.