In order to provide scientific basics for exploitation and sufficient application of Dendrobium officinale leaves resources, the phenol-sulfuric acid method was applied to determine the polysaccharide content. The monosaccharides were derivated by PMP and the derivatives were identified by HPLC-DAD-ESI-MS(n) and the contents of mannose and glucose were determined simultaneously. Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (2004A) was employed to generate the mean chromatogram and similarity analysis of the samples was carried out. The results demonstrated that polysaccharide content, monosaccharide compositions and composition ratio had an obvious difference between stems and leaves. The polysaccharide content of stems was higher than that of leaves. Monosaccharide composition in leaf was significantly different from that in stem. The polysaccharide from stems was composed of mannose and glucose, however the polysaccharide of leaves was acid heteropolysaccharide and was mainly composed of five monosaccharides, including mannose, galacturonic acid, glucose, galactose and arabinose. The similarity value of the 14 batches was above 0.9, indicating that similarity of fingerprints among different samples was high. The study can provide evidence for expanding the medicinal parts of D. officinale.