The invention relates to tulathromycin bacteria-free powder for injection and a preparation method of the tulathromycin bacteria-free powder, and belongs to the field of injection for animals. The preparation method comprises the following steps: (1), dissolving tulathromycin through anhydrous alcohol; (2), adding activated carbon, performing stirring until sufficient adsorption, and performing filtration so as to obtain a filtrate; (3), adding acid into the anhydrous alcohol, performing the filtration so as to obtain another filtrate, dropwise adding the filtrate into the filtrate obtained in the step (2), performing stirring so as to react, performing filtration so as to obtain a filter cake, after drying the filter cake under a reduced pressure, obtaining tulathromycin salt, namely the tulathromycin bacteria-free powder. The invention further provides the tulathromycin bacteria-free powder prepared through the preparation method and the application of the tulathromycin bacteria-free powder in preparation of needle injection of the tulathromycin bacteria-free powder. According to the invention, the problem that isomer exists in tulathromycin injection is solved, an isomer I with high activity is reserved, tulathromycin injection liquid is prepared into a single component, requirements for preparation are reduced, and the treatment effects are improved.