Mushrooms are full of nutrition and have beneficial properties for human health. Polysaccharides are the main component of edible and medicinal mushrooms, especially β-glucans, which have attracted much more attention for their complex structure and diverse biological activities. Among all the diverse medicinal activities of mushroom polysaccharides, antitumor and immune-enhancing activities are two excellent bioactivities that have much more potential and deserve application. Their bioactivities are highly dependent on their structural features, including molecular weight, monosaccharide composition, degree of branching, type and configuration of glycosidic bonds, substituent pattern, and chain conformation. This review summarizes the current method for obtaining polysaccharides from mushrooms, chemical characterizations of the structures and their roles in immune and antitumor activities. In addition, the methods for preparation of the polysaccharide derivatives and the potential medicinal clinical application are also discussed in this review, which may provide new guidance for mushroom polysaccharide development.