Polysaccharides isolated from medicinal plants and fungi exhibit multiple pharmacological activities. The biological activities of polysaccharides depend on their chemical characteristics. However, characterization of polysaccahrides is a challenge because of their complicated structure and macromolecular mass. In this review, chromatography in characterization of polysaccharides, including physicochemical characterization (purity, molecular mass, and distribution), structural characterization (constituent monosaccharide composition and the ratio, the features of glycosidic linkages), and fingerprint of polysaccharides (acidic and enzymatic hydrolysates), from medicinal plants and fungi were reviewed and discussed according to the publications collected in Web of Science since 2007. The perspective for characterization of polysaccharides has also been described.