Abstract: Determining the contents of amylose and amylopectin in tobacco with dual-wavelength spectrophotometry was new, although it has been widely applied to other crops. The measurement wavelengths of 512 and 596 nm for amylose and 560 and 695 nm for amylopectin were selected according to the principle of dual wavelength colorimetric, and the contents of amylose and amylopectin were calculated based on regression equations. The results showed that the contents of amylose and amylopectin in tobacco determined with dual-wavelength spectrophotometry were accurate, and the method was suitable for the determination of amylose and amylopectin in batches of tobacco samples.