Saffron stigmas are considered as one of the main sources for crocin extraction. Crocins have many therapeutic properties and applications in different industries such as food and cosmetic in which their extraction and purification will be useful commercially and clinically. This work presents a study on the partitioning of crocin in aqueous two-phase systems (ATPSs) composed of water, deep eutectic solvents (DESs), and acetonitrile (ACN). Two DESs based on choline chloride as the hydrogen bond acceptor (HBA) and urea and ethylene glycol as hydrogen bond donors (HBDs) were synthesized and considered as phase-forming components. Also, the effects of the hydrogen bonding, HBD species, weight percentages of DES, weight percentages of ACN, and temperature were investigated to evaluate the phase-forming ability of the ATPSs and the partitioning of crocin in these systems. It turned out that the choline chloride–urea/ACN system provides higher partition coefficients and a higher extraction efficiency of crocin under optimum conditions (40 wt % of DES, 50 wt % of ACN, and 25 °C). Moreover, the conformation of crocin in the choline chloride–urea/ACN system was explored using a UV–visible spectrophotometer (UV–vis) and Fourier transform infrared spectrometer (FT-IR), and no change was observed during the process. In the end, the UNIFAC model was applied to estimate the equilibrium data of both systems, in which the experimental data of two studied ATPSs were well described.