Carbon dots are often synthesized in the presence of a carbon source and passivating agents in which they are crucial for an enhanced fluorescence. The solvent choice and/or combination to be used in the synthesis of these nanoparticles can influence their surface chemical composition, morphology, and fluorescence properties. In this study, highly fluorescent carbon dots were synthesized using deep eutectic solvents of different compositions as green solvent media and doping agent. Resulting carbon dots were then separated by their hydrophilicity/hydrophobicity using a three-phase solvent system (water/ acetone/chloroform) and compared with traditional centrifugation-based separation method. Carbon dots with a size below 20 nm and quantum yield reaching 50% were obtained. Many properties of them including surface functional groups, optical, fluorescence, and electric properties were shown to be determined by the deep eutectic solvent composition.