In this study, two kinds of polysaccharides from leaves of Dendrobium officinale, namely DLP-1 and DLP-2, were obtained by hot water extraction, ethanol sedimentation, and chromatographic separation using DEAE-52 cellulose and Sephadex G-100 columns. They were composed of different monosaccharides and the content of monosaccharides varied significantly while DLP-1 (Mw 1.38 × 106 Da) was mainly composed of mannose (71.69%) and glucose (22.89%), and DLP-2 (Mw 1.93 × 106 Da) was constituted by rhamnose (35.05%), arabinose (24.12%), and galactose (25.65%). A triple-helical conformation was exhibited by both of them. The scanning electron microscope image of DLP-1 showed an irregular and large lamellar shape, as well as a smooth surface and a porous interior, illustrating they had an amorphous structure. In contrast, DLP-2 revealed a rough, loose, and uneven surface consisting of large sponge-like particles. Nuclear magnetic resonance analysis showed that (1→4)-β-D-Manp, (1→4)-β-D-Glcp, and (1→4)-2-O-acetyl-β-D-Manp were the main linkage types of DLP-1, whereas DLP-2 was constituted by a large amount of (1→4)-β-D-Manp, (1→4)-β-D-Glcp, and other residues. Besides, DLP-1 and DLP-2 stimulated the proliferation and phagocytic capacities of RAW 264.7 cells and improved the production of nitric oxide, interleukin-6, TNF-α, and IL-1β. These results proved that both DLP-1 and DLP-2 possessed excellent immunoregulatory bioactivities and could be functional food or adjuvant drug. PRACTICAL APPLICATIONS: The leaf of Dendrobium officinale is a by-product with huge biomass. The lack of systematic research on its chemical composition and pharmacologic effect, leading to a great waste of resources. In order to maximize the value of D. officinale, this study aimed to investigate the structural characteristics and immunologic effects of two polysaccharide fractions (DLP-1 and DLP-2) from D. officinale leaves, showing that DLP-1 and DLP-2 in D. officinale leaves could be used as anti-inflammatory agents to avoid wasting.