Abstract: The aqueous two-phase system of PEG/(NH4)2SO4， was used to purify hemoglobin(HB) in the initial salting out sample. The research investigated some parameters such as molecular weight of PEG, mass fraction of PEG and （NH4)2SO4, pH, temperature　which will influence partition ratio of hemoglobin. The results show that the best separation condition: wPEG400=20%, w(NH4)2SO4=20%, pH=7, temperature 20 ℃. The highest partition ratio can reach up to 65, the maximum value of extractive rate was 89.5%, and its purity was 80.5%.