An affinity extraction method combining an aqueous two-phase system and affinity ligand-bound fine magnetic particles is proposed as a novel protein separation technique. Polymer-coated magnetic particles, Eudragit-Mag, partitioned to the upper phase stably in three types of PEG-based aqueous two-phase systems. The phase separation time was shortened by the addition of the magnetic particles, and further reduced by applying a magnetic field. Human IgG was immobilized to Eudragit-Mag for the separation of staphylococcal protein A. The partition coefficient of protein A increased up to 35-fold when IgG-bound Eudragit-Mag was added to a PEG-phosphate aqueous two-phase system. The IgG-bound Eudragit-Mag was applied to the purification of protein A produced by recombinant Escherichia coil. In the separation, 90% of protein A added was selectively adsorbed to the affinity ligand, where the content of protein A was 89%. By using 3.5 M KSCN solution as an eluant, 39% of protein A was recovered with 49% of purity from a crude extract. In addition to the intrinsic advantages of aqueous two-phase extraction, the newly developed separation technique has advantages in the selective partitioning of the target protein, a shortening of phase separation time, and easy recovery and reuse of the ligand.