Aqueous two-phase systems (ATPS) are under focus, since those are considered efficient, environmentally friendly, and “biocompatible” separation processes, allowing the enzyme recovery with high purity levels. The common systems are based on polymers and salts and recently, to overcome the low polarity difference between the phases of the polymeric systems, ATPS based on ionic liquids (ILs) have been successfully applied on the field. However, the application of ILs as phase’s promoters in an ATPS normally requires the use of large concentrations of constituents, making the extraction process more expensive and less sustainable. The use of small quantities of ILs as adjuvants appears as an alternative to overcome this difficulty. This work discusses the use of imidazolium-based ILs as adjuvants (5wt%) in ATPS of polyethylene glycol with potassium phosphate buffer at pH 7, in the purification of a lipase produced by submerged fermentation by Bacillus sp. ITP-001. In here, a purification factor of 245 for the lipase was achieved with 1-hexyl-3-methyl imidazolium chloride. It was proved that the use of ATPS using ILs as adjuvants significantly enhances the purification performance when compared with the common polymeric and salt-ATPS, even when no pre-purification steps are implemented. The high purification factor was a consequence of the favorable interactions between the IL and the contaminant proteins that migrated for the PEG-rich phase, where the IL is preferentially concentrated, while the enzyme remains in the salt-rich phase.