Abstract: Effects of polyethylene glycol (PEG600 and PEG4000) on the ion exchange chromatographic behaviors of bovine hemoglobin (Hb) was studied. Pretreated bovine hemolysate was chromatographied with Q Sepharose Big Beads, and 0.5%～5.0% PEG600 or PEG4000 was added as a cosolvent. Consequently, the purity of Hb increased slightly, near to electrophoretical purity. The recovery of Hb was obviously higher than the control (76%), especially in the presence of PEG4000, which was over 90%. Bioactivity of Hb was well preserved in the process of chromatography after adding the cosolvent.The retention time of Hb in chromatography was not affected when PEG600 was added as the cosolvent, while it was longer in the presence of PEG4000 and the time increased as the concentration of PEG4000 improved. The results were discussed based on the theory of cosolvent.