•Seven polysaccharides (BGPs) are extracted from barley grass at three growth stages.•Different growth stages obviously affected physicochemical properties of these BGPs.•These BGPs had similar structural characteristics but different thermal stabilities.•BGP-Z12 and BGP-Z21 obtained at seedling stage showed more excellent bioactivities. Barley grass polysaccharides (BGPs) are some of the major bioactive constituents of barley (Hordeum vulgare L.) grass (BG). They exhibit favorable biological activities and health benefits. In this study, seven BGPs were extracted from BG, which was harvested at three different growth stages (e.g., seedling, tillering, and stem elongation), by alkaline-extraction method. Their physicochemical properties, structural characteristics, and biological activities were investigated and compared. Results demonstrated that the extraction yields, chemical compositions, monosaccharide constituents, and molecular weights of the seven BGPs obtained at different growth stages varied obviously. These BGPs had similar preliminary structural characteristics but different microstructures and thermal properties. Furthermore, the BGPs (BGP-Z12 and BGP-Z21) obtained at the seedling stage possessed stronger in vitro antioxidant potentials, cholic acid binding activity, and immunological activity than other BGPs. Therefore, these results indicated that that the seedling stage of BG was the preferable harvest time for preparing highly bioactive BGPs.