Polysaccharides are ubiquitous biomolecules found in nature that contain various biological and pharmacological activities that are employed in functional foods and therapeutic agents. Natural polysaccharides are obtained mainly by extraction and purification, which may serve as reliable procedures to enhance the quality and the yield of polysaccharide products. Moreover, structural analysis of polysaccharides proves to be promising and crucial for elucidating structure–activity relationships. Therefore, this report summarizes the recent developments and applications in extraction, separation, purification, and structural analysis of polysaccharides of plants and fungi.