Keratinase is widely applied in the bioconversion of feather waste to produce value-added bioproducts. The wide applications of industrial keratinase have urged the advancement in producing keratinase with high productivity and yields. The efficiency of aqueous two-phase electrophoresis system (ATPES) to recover extracellular Kytococcus sedentarius TWHKCO1 keratinase was investigated. The operation time of 10 min and voltage of 10 V were applied for the recovery of K. sedentarius TWHKCO1 keratinase with the PEG 1000/sulfate ATPES. Keratinase was recovered in the PEG-rich phase of the ATPES consists of 23% (w/w) PEG 1000, 18.5% (w/w) ammonium sulfate of pH 9, 20% (w/w) crude feedstock and with 3% (w/w) of NaCl added. Highest partition coefficient (K-e) of 4.63 +/- 0.17, recovery yield (Y-T) of 78.75% +/- 0.61, and purification fold (PFT) of 3.89 +/- 0.84 were obtained in the optimal ATPES coupled with cathode electrode in top phase and anode electrode in the bottom phase. The introduction of electric field to the ATPS has enhanced the selectivity (S) of the system towards the extracellular keratinase by two-fold from 2.79 +/- 0.46 to 5.68 +/- 0.43, demonstrating the efficiency of ATPES in separating the keratinase from crude feedstock when compared to conventional two-phase system.