Cytochrome c is a small water-soluble protein that is abundantly found in the mitochondrial intermembrane space of microorganism, plants and mammalians. Ionic liquids (ILs)–based aqueous two-phase electrophoresis system (ATPES) was introduced in this study to investigate the partition efficiency of cytochrome c to facilitate subsequent development of two-phase electrophoresis for the separation of cytochrome c from microbial fermentation. The 1-Hexyl-3-methylimidazolium bromide, (C 6 mim)Br and potassium citrate salt were selected as the phase-forming components. Effects of phase composition; position of electrodes; pH and addition of neutral salt on the partition efficiency of cytochrome c in the ATPES were evaluated. Highest partition coefficient ( K  = 179.12 ± 0.82) and yield of cytochrome c in top phase ( Y T  = 99.63% ± 0.00) were recorded with IL/salt ATPES composed of 30% (w/w) (C 6 mim)Br and 20% (w/w) potassium citrate salt of pH 7 and 3.0% (w/w) NaCl addition with anode at the bottom phase and cathode at the top phase. The SDS-PAGE profile revealed that cytochrome c with a molecular weight of 12 kDa was preferably partitioned to the IL-rich top phase. Present findings suggested that the single-step ATPES is a potential separation approach for the recovery of cytochrome c from microbial fermentation. Graphical Abstract