Pathogenic mycobacteria, including the causative agents of tuberculosis, are responsible for considerable morbidity and mortality worldwide. The availability of a laboratory method that can distinguish between two groups of pathogens; Mycobacterium tuberculosis complex (MTC or MTBC), nontuberculous mycobacteria (NTM) and those who have only had a history of dealing with non-pathogenic or recently vaccinated individuals is of great importance. In this regard, different strains of mycobacteria were cultured in the Dorset-Henley liquid medium and the inactivation of the cultures is carried out by the heat. The bacteria were separated from the liquid medium containing secreted proteins with EKS filters. Low molecular weight proteins in Mycobacterium tuberculosis precipitated with ammonium sulfate were purified by Sephadex-G50 gel chromatography and crude antigens in other mycobacteria precipitated with TCA. Protein concentrations determined with lowry protein assay, antigens coated on the ELISA plate and the results were analyzed with SPSS software and investigated. In this study, all antigens had more than 92% detection ability in healthy livestock in the ELISA method. The highest specificity was related to ESAT-6/CFP10 and M. avium subsp. Paratuberculosis antigens were 83.66% and 95.83% respectively and the highest efficiency of diagnostic tests were over 83% concerning these two antigens. It concluded that the two antigens ESAT-6/CFP10 and M. avium subsp. Paratuberculosis are suitable candidates for the design of the diagnostic ELISA system due to their sensitivity, specificity and efficiency and also reliable detection of healthy livestock from sensitized livestock.