The invention relates to the technical field of poultry antibody detection reagents, particularly to a mycoplasma gallisepticum antibody detection reagent, a preparation method and application thereof. According to the invention, a mycoplasma gallisepticum culture solution is subjected to a series of treatment on, inactivating is performed, the inactivated antigen bacteria solution and a freeze-drying protective agent are uniformly mixed, and freeze drying is performed to obtain the mycoplasma gallisepticum antibody detection reagent; the reagent can be directly used for a hemagglutination inhibition test; a plate agglutination test can be carried out by adding any one coloring agent of methyl violet, amber red and crystal violet into the reagent containing 4-8HA unit of antigen; the hemagglutination value of the detection reagent reaches 26; and the detection reagent not only improves the hemagglutination titer of the mycoplasma gallisepticum antigen, but also has two purposes, is stable, sensitive, strong in specificity, long in storage life, simple to operate, convenient and fast, does not need special equipment and instruments, and can be used for efficacy test, epidemiologicalinvestigation, clinical sample detection and the like of mycoplasma gallisepticum vaccines.