To evaluate the specificity of single intradermal cervical tuberculin (SICT) of bovine purified protein derivative (PPD) in bovine tuberculosis detection, a total of 118 tissue specimens from 54 cows with positive reaction were collected and subjected to the detection of Mycobacterium. Acid-fast bacteria strains were isolated from 14 cows and Mycobacterium strains were isolated from 10 cows. Mycobacterium bovis (M. bovis) was isolated from one cow. Sixteen acid-fast bacteria strains were isolated from 118 tissue samples and 12 strains were identified as Mycobacterium. The isolation rate of Mycobacterium was 10.2% (12/118). One strain was identified as M. bovis and the others were nontuberculous Mycobacteria (NTM), such as M. avium and M. terrace. M. bovis accounted for 8.3% (1/12) and NTM accounted for 91.7% (11/12) of all Mycobacteria respectively. The results of pathogen detection indicated a low isolation rate of M. bovis by SICT method. To ensure the accuracy of SICT, it is necessary to use more than one method to verify the results. The culture and species identification by using rapid detection techniques are recommended to be introduced into the program for tuberculosis elimination, which could improve the specificity of tuberculosis detection.