The application discloses a preparation method of a specific yolk antibody resisting mycoplasma synoviae diseases. The preparation method comprises the following steps of recovering mycoplasma synoviae strains, performing propagation culture for three generations, performing sufficient inactivation, performing centrifuging, and taking precipitate so as to obtain a mycoplasma synoviae antigen; performing mixed emulsification on a mycoplasma synoviae antigen solution and a white oil adjuvant, performing basic immunization on each hen, and performing injection for 0.5ml; and after the basic immunization, performing strengthened immunization twice, wherein an immunization method and the dosage are the same as those of the basic immunization, after the first-time strengthened immunization, beginning to collect immunized eggs, disinfecting the obtained immunized eggs with alcohol, breaking shells, collecting yolk, and performing purifying to obtain the specific yolk antibody. The specific yolk antibody resisting mycoplasma synoviae diseases, prepared by the preparation method can effectively prevent and treat the mycoplasma synoviae diseases. The specific yolk antibody is easy in mass production and low in cost, and an obtained product is a safe preparation resisting the mycoplasma synoviae diseases.