The test results of the bovine tuberculin skin test (TST), acid-fast stain test, ELISA test and bovine IFN-γ- ELISA diagnosis test respectively were compared for detection of tuberculosis of them, three stimulators, detected by IFN-γ-ELISA including bovine PPD, avian PPD and bovine PPD at affinity chromatography were compared in exploration for application of purification of diagnostic antigen in the IFN-γ-ELISA method. Using acid-fast staining as the standard, the sensitivity of IFN-γ-ELISA detection reached 83.33%(5/6), and its specificity reached 98.14%(53/54), the consistency reached 98.33% (58/60). As the other result, acid-fast stain detected a positive sera with high OD value and produced a positive result using bovine IFN-γ-ELISA in which the antigen was not purified at affinity chromatography, but the result was negative with low OD value using bovine IFN-γ-ELIS in which the antigen was purified at affinity chromatography. In conclusion, the miscarriage of justice caused by infected with Mycobacterium bovis and environmental Mycobacterium simultaneouly could be prevented using bovine IFN-γ-ELISA in which the antigen was purified at affinity chromatography. So this method has great potential to replace TST for development and application prospects in China.