Background: The metabolic compounds from tobacco were investigated to posses various biological activities, such as antioxidant, antimicrobial and neuroprotective activities. Materials and Methods: Polysaccharides were isolated from the ultrasound-assisted extraction of flue-cured tobacco with hot water. The purified polysaccharides were analyzed by GC-MS, FT-IR, and TGA (Thermogravimetric analysis). The antioxidant activities of purified polysaccharides were evaluated in vitro. Results: Two polysaccharides (Fr-I and Fr-II) were isolated and purified. The analysis of monosaccharide composition in the polysaccharide by GC/MS revealed that Fr-I and Fr-II were heteropolysaccharides. Fr-I consisted of D-mannose and galactose, ribose and arabinose, while Fr-II was mainly composed of glucose, allose, galactose and mannose. Thermo gravimetric analysis (TGA) indicated that the degradation temperature (Td) of the Fr-I (241 °C) was higher than that of Fr-II (220 °C). Both samples showed inhibitory effects on OH (hydroxyl) and DPPH (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) radical in a concentration-dependent manner. Comparing Fr-I with Fr-II, the latter has a strong scavenging ability. Conclusion: Both polysaccharide fractions showed significant antioxidant effects. Various factors influenced the antioxidant activity of polysaccharides.