Crude water-soluble polysaccharides were extracted from the stem of Dendrobium nobile Lindl. using boiling-water. The polysaccharides were successively purified by chromatography on DEAE-cellulose and Sephadex G-200 column, giving four major polysaccharide fractions termed DNP1-1, DNP2-1, DNP3-1, DNP4-2. The gel permeation chromatography (GPC) analysis showed that the average molecular weight (Mw) of polysaccharides (DNP1-1, DNP2-1, DNP3-1, DNP4-2) were approximately 136 kDa, 27.7 kDa, 11.8 kDa and 11.4 kDa, respectively. Monosaccharides analysis revealed the dominance of mannose, glucose, galactose, and smaller amounts of rhamnose, arabinose and xylose in the four polysaccharides. The evaluation of antioxidant activity suggested that DNP4-2 had good potential for scavenging activity of ABTS radical, and higher scavenging activity of hydroxyl radical and DPPH radical than the other polysaccharides, and should be explored as a novel potential antioxidant.