A series of ethylene oxide (EO)-propylene oxide (PO) random co-polymers (EOPO) were used to form aqueous two-phase systems (ATPS) with ammonium sulfate. Effects of EOPO's properties on the phase separation behaviors and on the partition of cephalexin and 7-aminodesacetoxicephalosporanic acid (7-ADCA) in ATPS were investigated. Both the molar mass and molar ratio of EO to PO of EOPO could greatly influence partition behaviors of cephalexin and 7-ADCA as well as the binodal curve of ATPS. With the increase of molar mass of co-polymer or the decrease of molar ratio of EO to PO, the critical point of ATPS decreased, the binodal curve became more asymmetry, and both cephalexin and 7-ADCA followed the same tendency to partition into the polymer-poor bottom phase. The experimental results shows that it is feasible to partition cephalexin and 7-ADCA in either the polymer-rich top phase or the polymer-poor bottom phase by choosing a specific phase-forming EOPO.