A deep eutectic solvent (DES)-based aqueous biphasic system (ABS) denotes a clean and highly biocompatible separation and purification technology, but the explanations of the phase formation mechanism and phase behavior of a DES-based ABS are incomplete. To improve the study of the formation mechanism of a DES-based ABS, a new ABS composed of n-propanol (NPA) and DES with betaine as the hydrogen-bond acceptor (HBA) and polyol as the hydrogen-bond donor (HBD) was formed. The phase formation mechanism of this new ABS and the stability of the DES in the ABS were studied. Betaine and three polyols are hydrophilic (log KOW < 0), whereas NPA is relatively hydrophobic (log KOW > 0). Two phases were formed, because the two components of DES were incompatible with the NPA. The molar ratio of the HBA and HBD in the two phases differed, but in the bottom phase of this ABS, the stoichiometric ratio of HBA and HBD was the same as their initial ratio. Under certain conditions, this novel ABS was considered a ternary-like system. The combination of a series of factors, such as HBD hydrophilicity, molar volume, molecular weight, and initial molar ratio of HBA and HBD, affected the final stoichiometric ratio of HBA and HBD in the top phase.