Many unknown impurities were produced in the synthesis and degradation of tulathromycin, which was difficult to sep. and analyze. The impurities in the synthesis of tulathromycin were eluted and separated by preparative liquid chromatog. The unknown impurities were distilled under reduced pressure, extracted and desalted, and the impurity tulathromycin-D was obtained by rotary evaporation and concentration of the organic phase. The structure of the product was confirmed by NMR and HRMS, which laid a good foundation for the study of unknown impurities of tulathromycin.