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In the present work, we developed an acousto-optic tunable fi lter near infrared (AOTF-NIR) refl ectance spectroscopy method for determining tobacco constituents. The NIR spectra of 443 tobacco samples were collected using an AOTF-NIR spectrometer. Calibration models for total sugar, reducing sugar, nicotine and potassium in tobacco were built by partial least squares (PLS) regression for which regression R 2 were 0.984, 0.987, 0.988 and 0.920, respectively and the average relative standard deviations between the predicted values and the measured values were 2.68%, 2.88%, 4.18% and 8.38%, respectively. The results indicate that the AOTF-NIR technique is feasible for the analysis of tobacco constituents and provides a foundation for on-line monitoring in the tobacco process. To our knowledge, this is the fi rst attempt to analyse tobacco constituents with AOTF-NIR spectroscopy.