Abstract Polysaccharides, with an extensive distribution in natural products, represent a group of natural bioactive substances having widespread applications in health-care food products and as biomaterials. Devising an efficient system for the separation and purification of polysaccharides from natural sources, hence, is of utmost importance in the widespread applicability and feasibility of research for the development of polysaccharide-based products. High-speed counter-current chromatography (HSCCC) is a continuous liquid–liquid partitioning chromatography with the ability to support a high loading amount and crude material treatment. Due to its flexible two-phase solvent system, HSCCC has been successfully used in the separation of many natural products. Based on HSCCC unique advantages over general column chromatography and its enhanced superiority in this regard when coupled to aqueous two-phase system (ATPS), this review summarizes the separation and purification of various bioactive polysaccharides through HSCCC and its coupling to ATPS as an aid in future research in this direction.