Focused on developing a biocompatible and ecofriendly extraction platform, aqueous two-phase systems (ATPS) containing betaine-xylitol deep eutectic solvent (DES), three smallmolecular alcohols (1-propanol, 2-propanol, and tert-butanol), and water were investigated here. The effect of alcohol properties on the efficiency to form ATPS with aqueous solution of DES was studied at 298.15 K and atmospheric pressure. The results suggested that the formation of the ATPS was attributed to the incompatibility between the DES and alcohol in aqueous medium, allowing the increase in the biphasic region using tert-butanol with the highest hydrophobicity among three alcohols. Combined with Karl Fischer titration, the high-performance liquid chromatography (HPLC) analysis was used to evaluate the tie-lines of the ATPS, and it was found that DES components were enriched in the lower phase while alcohol was transferred preferentially to the upper phase. Additionally, the increase in the concentrations of ATPS phase compositions was responsible for the increase in the length of tie-lines, which led to a higher driven force for phase separation. The detailed experimental data were highly correlated by the NRTL model based on a low root-mean-square deviation (≤1.79%). Moreover, five biomolecules (gallic acid, syringic acid, quercetin, kaempferol, and isorhamnetin) were used as probes to determine the extraction ability of the studied DES/alcohol ATPS. This work may lay a promising foundation for the diversity development of the ATPS.