A new method of using ethanol-K2HPO4 aqueous two-phase system for extracting spiramycin was established. The optimum equation suitable for bimodal data fitting of the system was selected. The influence of K2HPO4 mass fraction, ethanol mass fraction, spiramycin concentration, extraction temperature and pH on the partition coefficient and extraction efficiency was investigated. The experimental results showed that the extraction process of spiramycin was spontaneous, endothermic and entropy increasing. Under the condition of K2HPO4 mass fraction of 20%, ethanol mass fraction of 16%, extraction temperature of 25℃, and pH of 9.2~9.5, the partition coefficient and extraction efficiency of spiramycin were above 36.66 and 97.11%, respectively. When pH = 9.5, the partition coefficient reached 47.52 and the extraction efficiency reached 97.97%. The purification factor and extraction efficiency of spiramycin using ethanol-K2HPO4 aqueous two-phase extraction were high. The phase-forming materials ethanol and K2HPO4 can be recycled for reuse, avoiding secondary pollution. A new method was provided for the treatment of isovalerylspiramycin production wastewater containing spiramycin.