Dimeric bovine hemoglobin (Hb) tetramers were prepared by a one-step solid phase adsorption method. Briefly, Hb was absorbed by the solid phase, Q Sepharose Fast Flow media, followed by reaction with the glutaraldehyde and elution procedure. Then, dimeric bovine Hb tetramers were formed and purified from Hb tetramers by anion-exchange chromatography based on Protein-Pak DEAE 8HR. The dimeric Hb tetramer showed a P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2%, and Hill coefficient of 1.72. The number of Bohr protons released for dimeric Hb tetramers was 0.59 H/tetramer, which was 39% of that of native bovine Hb. The number of chloride ions released on oxygenation was 0.60/tetramer for dimeric Hb tetramers, which was 46% of that of native bovine Hb.