A crude polysaccharide fraction (cDOP) has been determined to be the characteristic marker of Dendrobium officinale, an expensive tea material in Asia, but its chemistry and bioactivity have not been studied. In work reported here, cDOP was destarched (DOP, 90% yield) and separated into two subfraction polysaccharides, DOPa and DOPb, which were characterized by monosaccharide composition and methylation analyses and spectral analyses (FT-IR and 1H and 13C NMR). Both are composed of mannose and glucose at similar ratios and have a similar structure with a backbone of 1,4-linked β-d-mannopyranosyl and β-d-glucopyranosyl residues. Significant differences were observed only in their molecular weights. Bioassay using mouse macrophage cell line RAW264.7 indicated that DOP and its two subfractions enhance cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. They also induced the proliferation of lymphocytes alone and with mitogens. DOPa and DOPb are thus proven to be major, active polysaccharide markers of D. officinale.