The aim of this work is the synthesis of deep eutectic solvents (DESs) based on choline chloride (ChCl) and sugars to form new aqueous two-phase systems (ATPS) with acetonitrile (ACN) followed by application in vanillin partitioning. The binodal curves were not altered by the different DESs and have a smaller biphasic area than ChCl and sugars. However, the ATPS based on DES and ACN provided the vanillin partition to top phase (Kvan > 10.07) and 89% of recovery, higher than ATPS based on ACN and ChCl and similar to ATPS based on ACN and sugars. The ATPS (ACN + DES/ChCl-Gli) was analyzed whether it was ternary or quaternary, revealing to be ternary.