A pH-responsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogel was prepared by an aqueous dispersion polymerization process. The number of carboxylic groups distributed on the surface of the hydrogel was found to be 1.89 mmol g(-1). The hydrogel exhibits excellent binding selectivity to the protein species of interest within certain pH regions. In the present work, it was applied to the selective isolation of hemoglobin from human whole blood. Some important experimental parameters governing the sorption property, including the pH of sample solution, the ionic strength and sorption time, were investigated. Using 2.0 mg of the hydrogel as a sorption medium and suitable acidity of pH 6.0, a sorption efficiency of 93% was achieved for 200 microg mL(-1) of hemoglobin in 1.0 mL of sample solution. Afterwards, the absorbed hemoglobin could be readily recovered using 1.0 mL of Tris-HCl buffer (pH 8.6, 0.01 mol L(-1)), giving rise to recovery of ca. 76%. The practical applicability of this system was further demonstrated by processing human whole blood for the isolation of hemoglobin, and satisfactory results were obtained by assay with SDS-PAGE.