The present invention provides a mycoplasma vaccine, its preparation and application thereof. The foregoing mycoplasma vaccine comprises inactivated Mycoplasma hyorhinis ATIT-7 only or the mixture of inactivated Mycoplasma hyorhinis ATIT-7 and inactivated Mycoplasma hyopneumoniae, which effectively prevents the infection of swine enzootic pneumonia in pigs.