Aqueous two-phase systems (ATPSs) provide a versatile method for the extraction, separation, purification, and enrichment of various biomolecules and other valuable materials. Due to their high water content, the interfacial tension in ATPSs is significantly lower than in water–organic biphasic systems, facilitating easy transfer between phases and providing a gentle environment that preserves the integrity of sensitive biomolecules. ATPSs provide substantial sustainability benefits in biomolecule extraction, such as reduced energy consumption, lower environmental impact, and improved cost efficiency. The use of carbohydrates in ATPSs offers additional advantages resulting from their biodegradability, nontoxicity, and renewability, making these systems particularly attractive for sustainable biomolecule purification and extraction processes. Compared with salting-out methods, the sugaring-out technique is simpler, more stable, and cost effective and reduces equipment corrosion. Carbohydrate-based ATPSs cut energy use by 30–60% and generate minimal waste, resulting in lower carbon emissions and reduced hazardous waste. These systems also lower raw material and waste management costs by 30–60%, making ATPS an environmentally and economically advantageous option for industrial applications. Carbohydrates can also function as both phase formers and chiral selectors, creating chiral ATPSs useful for enantioseparations. This perspective explores the fundamental and practical aspects of polymer–carbohydrate and ionic liquid (IL)–carbohydrate ATPSs, discussing the sugars, ILs, and polymers used, the methods for determining phase diagrams, and the various target materials extracted and purified using these systems. The molecular mechanisms underlying the formation of these ATPSs and the impact of different parameters on their phase behavior are also examined. Thermodynamically, the formation of ATPSs is driven by an increase in the entropy. The results show that more hydrophilic carbohydrates, when paired with more hydrophobic polymers or ILs, have a stronger tendency to form ATPSs. This suggests a mechanism in which the hydrophobic polymer or IL is effectively “sugared-out” by the carbohydrate, promoting phase separation. The perspective concludes with an evaluation of the applications of these systems in the extraction, separation, and purification of diverse materials.
