Deep eutectic solvents (DESs) are regarded as a new class of green solvents because of their unique properties such as easy synthesis, low cost, environmental friendliness, low volatility, high dissolution power, high biodegradability, and feasibility of structural design. DESs have been widely applied for the separation of mixtures as alternatives to conventional solvents. A DES usually consists of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). HBAs include amides, thiourea, amines, imidazole, azole, alcohols, acids and phenol. HBAs include quaternary ammonium salts, quaternary phosphonium salts, imidazolium-based salts, dication based salts, inner salts, and molecular imidazole and its analogues. Therefore, there are numerous DESs available for use in different applications. With an in-depth understanding of the common and novel properties of DESs, researchers have prepared and applied DESs to various types of separations. We first introduce the composition of DESs, including various HBDs and HBAs frequently used in the literature. Second, the properties of DESs, including phase diagrams, melting points, density, viscosity, and conductivity, are summarized. Third, recent applications of DESs in the separation of mixtures are reviewed, including the absorption of acidic gases (CO2, SO2 and H2S), the extraction of bioactive compounds, extraction of sulfur-and nitrogen-containing compounds from fuel oils, extraction of phenolic compounds from oils, separation of mixtures of aromatic and aliphatic compounds, separation of alcohol and water mixtures, removal of glycerol from biodiesel, separation of alcohol and ester mixtures, removal of radioactive nuclear contaminants, and separation of isomer mixtures of benzene carboxylic acids. DESs are used in two ways for the separation of mixtures. (1) A DES prepared in advance is used as a solvent to separate a component from a mixture by selective dissolution or absorption of specific compound(s), such as the absorption of SO2 using betaine+ethylene glycol DES. Here, DESs are used like traditional solvents. (2) A DES is formed in situ during the separation of mixtures by adding a HBA to a mixture containing one or more HBDs, such as the removal of phenol from an oil mixture using choline chloride, where a phenol+choline chloride DES is formed during the separation process and the formed DES does not dissolve in the oil phase. Although various DESs have been broadly developed for the separation of mixtures, research continues in the field of DESs, including analysis of the physicochemical properties of DES, especially during extraction or absorption, development of functional DESs for high-efficiency separations, development of efficient methods to regenerate DESs, and combined use of DESs with other techniques to improve separation processes. This article describes general trends in the development of DESs for separation and evaluates the problematic or challenging aspects of DESs in the separation of mixtures.