Membrane separation technology, owing to its low energy consumption, operational simplicity, ease of control and scale-up, has gained increasing interest. The rapid development requires highly selective membranes with high through rates and chemical/mechanical stability. Chitosan, a relatively inert biomaterial with film forming ability, has been rapidly recognised for its potential in separation and purification technology in recent decades. Because of its hydrophilicity, cationicity, biocompatibility, ease of modification, remarkable affinity to dyes, metals and proteins, chitosan membranes have become a promising candidate for several applications. In the present review, the pervaporation of azeotropic mixtures and protein purification and separation of gaseous mixtures using chitosan membranes are discussed. The general overview of the preparation of different chitosan membranes, its capabilities in the separation of various solvents, gases and biomacromolecules are presented. Finally, the opportunities, challenges and perspectives in these areas are discussed.