Beta-carotene was isolated from discarded tobacco leaves by column chromatography using petroleum ether as eluent. Its structure was characterized by 1H-NMR and 13C-NMR spectra. The purity of the beta-carotene was determined to be 98% by a high performance liquid chromatographic (HPLC) method which was demonstrated to be accurate and reproducible. Infra-red spectrum and melting point of the isolated sample were also characterized in agreement with those reported in literature. The overall yield of 80%, the high purity and the easiness to recycle as the results of only a single solvent employed, would make the method an important reference for future research of beta-carotene and a favorable technique for future development.