The invention provides a method for separating and purifying tulathromycin D. The method comprises the following steps: eluting a to-be-separated substance in a chromatographic column by using a mobile phase by adopting high performance liquid chromatography, performing reduced pressure distillation on qualified fractions, extracting and desalting, and performing reduced pressure concentration onan organic layer until the organic layer is dried. According to the method disclosed by the invention, the qualified fractions are treated before extraction, the qualified fractions are subjected to high-pressure homogenization or ultrasonic grinding, the particle size is reduced, the pH value is regulated, activated carbon for injection is added, stirring and decarburization are performed, filtration is performed by virtue of a filter membrane, the apparent distribution volume is improved, and impurities in the fractions are well separated. According to the separation method, the impurities produced in the synthesis and degradation process of the tulathromycin can be effectively separated, the production batch is stable, the quality is reliable, the production cycle is short, the single preparation yield is high, the impurity quality control requirements are met, and a good foundation is laid for research of unknown impurities of the tulathromycin.