Chondroitin sulfate (CS), a natural anionic mucopolysaccharide, belonging to the glycosaminoglycan family, acts as the primary element of the extracellular matrix (ECM) of diverse organisms. It comprises repeating units of disaccharides possessing β-1,3-linked N-acetyl galactosamine (GalNAc), and β-1,4-linked D-glucuronic acid (GlcA), and exhibits antitumor, anti-inflammatory, anti-coagulant, anti-oxidant, and anti-thrombogenic activities. It is a naturally acquired bio-macromolecule with beneficial properties, such as biocompatibility, biodegradability, and immensely low toxicity, making it the center of attention in developing biomaterials for various biomedical applications. The authors have discussed the structure, unique properties, and extraction source of CS in the initial section of this review. Further, the current investigations on applications of CS-based composites in various biomedical fields, focusing on delivering active pharmaceutical compounds, tissue engineering, and wound healing, are discussed critically. In addition, the manuscript throws light on preclinical and clinical studies associated with CS composites. A short section on Chondroitinase ABC has also been canvassed. Finally, this review emphasizes the current challenges and prospects of CS in various biomedical fields.