To separate spiramycin from the crude sample of bitespiramycin, the optimal extraction conditions in single stage extraction had been obtained as follows: the temperature was 30-40 degrees C, the concentration of phosphate solution was 0.3-0.4 wt.%, and the volumetric ratio of ester/aqueous was 2-3. A three-stage counter-current extraction technique had been utilized to improve extraction efficiency. The removal rate of spiramycin and the retention rate of bitespiramycin were as high as 79.14% and 92.41%, respectively. A theoretical model of three-stage counter-current extraction was introduced to calculate the results of the extraction and fitted the experimental data with satisfactory accuracy.