A polymer-salt aqueous two-phase system based on polyethylene glycol and sulfates was applied to explore the partition behavior of spiramycin. Binodal curves were measured by the turbidimetric titration method. The influence of temperature, salt concentration, and pH on the distribution behavior was investigated in detail by determining the partition coefficient and extraction efficiency. As pH and salt concentration increase, the partition coefficients increase accordingly. It was found that pH displays a significant influence on the partition behavior. The partition coefficient and extraction efficiency can reach 42.46 and 97.8%, respectively, at pH 9.0 and 15.80% (w/w) salt concentration.