Partitioning of bovine serum albumin (BSA) in an aqueous double countercurrent distribution (DCCD) using a three-phase system was studied and compared with countercurrent distribution using a two-phase system. The two-phase system was composed of Dextran and Polyethylene glycol (PEG) solutions, and the three-phase system consisted of Dextran, Ficoll, and PEG solutions. In the double countercurrent distribution experiment, BSA was partitioned from the stationary bottom phase into the moving middle and top phases. Two peaks resulted from these transfers. A mathematical model that predicts peak positions and shapes based on the partitioning behavior of the solute is provided. Based on model predictions, the DCCD method has potential extraction selectivity and recovery advantages over the traditional CCD method.