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 de-starched (DOP, 90% yield), and separated into two polysaccharides, DOP-1 and DOP-2, which were characterized by monosaccharide composition and methylation analyses, and spectral analyses (FT-IR and 1 H and 13 C 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 RAW 264.7 indicated that DOP and its two sub-fractions enhance cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. They also induced the proliferation of lymphocytes alone and with mitogens. DOP-1 and DOP-2 are thus proven to be major, active polysaccharide markers of D. officinale.