[Background] Bovine tuberculosis is a second-class animal disease in China, and it was listed as a legally reported animal disease by Word Organisation for Animal Health (OIE). Cattle are mainly infected by aerosols produced by respiratory secretions and coughs in Mycobacterium bovis-infected cattle; people are mainly infected with meat or milk from diseased cattle that have not been treated with high temperature. Therefore, the rapid detection of suspected diseased milk or slaughter tissue samples by pathogen PCR detection can minimize the economic loss of dairy farming industry, which is of great significance. [Objective] To study and determine the suitable PCR amplification primers and parameters of Mycobacterium bovis, providing reference for rapid and accurate diagnosis of Bovine tuberculosis. [Methods] For the five pairs of PCR primers reported, the appropriate annealing temperature (Tm) was determined by touch down PCR; to determine the sensitivity of PCR methods with different primers, we used the genomic DNA of the Mycobacterium bovis C68001 strain (the domestic strain for bovine tuberculin production) and the artificial liquid with different bacterial contents in simulate clinical samples (lymph nodes, lungs, and milk); and then 6 common bacteria infecting bovines (B. abortus 2308, B. melitensis Rev.1, M. bovis C68001 and AN5, M. avium C68202, M. paratuberculosis C68681 and M. intracellulare) were uesed to determine the specificity. [Results] All primers contained the target band at 53?63 °C, and the best suitable Tm was 60 °C. The primers No. 1 and No. 3 had the highest sensitivity detecting nucleic acid of C68001, reaching 10?10 ng/μL; followed by No. 2 and No.5, up to 10?5 ng/μL. For artificially simulated infection samples, primers No. 1, 3, and 4 were most sensitive to detection in lymph nodes and lungs, followed by No. 2; and primers No. 2, No. 3, No. 4, and No. 5 were the most sensitive to milk samples. For specificity tests, primers 2 and 5 have better specificity which can detect significant M. bovis specific bands. It is weak when detecting M. avium that do not normally cause bovine tuberculosis but interfere with immunological diagnosis, and it had no bands in brucella, M. bovis, M. paratuberculosis and M. intracellulare. [Conclusion] The PCR method with primer No. 2 had the best sensitivity and specificty, so it is suitable for rapid and accurate diagnosis of bovine tuberculosis.