AbstractIn this study, Dendrobium officinale polysaccharide (named DOPS‐1) was isolated from the stems of Dendrobium officinale by hot‐water extraction and purified by using Sephadex G‐150 column chromatography. The structural characterization, antioxidant and cytotoxic activity were carried out. Based on the results of HPLC, GC, Congo red experiment, together with periodate oxidation, Smith degradation, SEM, FT‐IR, and NMR spectral analysis, it expressed that DOPS‐1 was largely composed of mannose, glucose and galacturonic acid in a molar ratio of 3.2 : 1.3 : 1. The molecular weight of DOPS‐1 was 1530 kDa and the main chain was composed of (1→4)‐β‐D‐Glcp, (1→4)‐β‐D‐Manp and 2‐O‐acetyl‐(1→4)‐β‐D‐Manp. The measurement results of antioxidant activity showed that DOPS‐1 had the strong scavenging activities on hydroxyl radicals, DPPH radicals and superoxide radicals and the high reducing ability in vitro. Moreover, DOPS‐1 was cytotoxic to all three human cancer cells of MDA‐MB‐231, A549 and HepG2.