Fungi are a rich source of bioactive polysaccharides with demonstrated nutritional and health-promoting properties, making them valuable for applications in the food, pharmaceutical, cosmetic, and materials science industries. The degree of polysaccharide purity required varies depending on the intended application, necessitating a clear understanding of appropriate extraction and purification techniques. Moreover, due to the structural complexity and heterogeneity of fungal polysaccharides, it is essential to characterize their composition and structure accurately. This chapter presents a comprehensive protocol for the isolation, purification, and characterization of crude polysaccharides from fungal biomass, enabling researchers to obtain materials with varying levels of purity. Additionally, it describes analytical procedures for quantifying total carbohydrate content using the phenol-sulfuric acid method, measuring acetyl group content for chitin estimation, and determining monosaccharide composition. These protocols are critical for understanding structure-function relationships, bioactivity assessments, and tailoring their physicochemical properties for applications in biomedical, nutraceutical, and biopolymer technologies.