In general, the methodologies for the preparation of carbon dots (CDs) lead to the formation of nanostructures with size and surface chemistry heterogeneity. Because the electronic and optical properties of these nanoparticles are directly associated with these properties, the development of purification and selection strategies is essential. Herein, we report a systematic study of the spontaneous partition and separation of highly oxidized carbon dots (OCDs) prepared by the dehydration and oxidation reactions of cotton cellulose in aqueous two-phase systems (ATPSs) based on polymer-salt pairs. The partition of the CDs was investigated in different ATPSs in which the effects of the cations and anions of the salts, molecular mass and nature of the polymer, tie-line length, initial pH, and surface modification of the nanoparticles on the partition coefficient (K) were evaluated. The results showed that the best separation occurred with a system consisting of PEO1500 + lithium sulfate + water using reduced CDs with hydrazine. Alternatively, the lowest value of K, 0.79, was obtained for a poly(ethylene oxide) PEO1500 + sodium tartrate + water system with pH = 6 using OCDs. The detailed analyses of the top and bottom phases of the systems with fluorescence and ultraviolet-visible spectroscopy showed that ATPSs are capable, in addition to partitioning, of separating the nanoparticles with different optical properties, which are directly associated with the surface properties and particle sizes. We believe that the presented methodology is an alternative, practical, fast, and potentially scalable technique for the separation of carbon nanostructures with different optical properties.