Tuberculosis (TB), a contagious disease mainly caused by Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), and Mycobacterium caprae (M. caprae), poses a major global threat to the health of humans and many species of animals. Developing an ante-mortem detection technique for different species would be of significance in improving the surveillance employing a One Health strategy. To achieve this goal, a universal indirect ELISA was established for serologically detecting Mycobacterium tuberculosis complex infection for multiple live hosts by using a fusion protein of MPB70, MPB83, ESAT6, and CFP10 common in M. tb, M. bovis, and M. caprae as the coating antigen (MMEC) and HRP-labeled fusion protein A and G as a secondary antibody. After testing the known positive and negative sera, the receiver operating characteristic curves were constructed to decide the cut-off values. Then, the diagnostic sensitivity and specificity of MMEC/AG-iELISA were determined as 100.00% (95% CI: 96.90%, 100.00%) and 100.00% (95% CI: 98.44%, 100.00%) for M. bovis infection of cattle, 100.00% (95% CI: 95.00%, 100.00%) and 100.0% (95% CI: 96.80%, 100.00%) for M. bovis infection of sheep, 90.74% (95% CI: 80.09%, 95.98%) and 98.63% (95% CI: 95.14%, 99.76%) for M. bovis infection of cervids, 100.00% (95% CI: 15.81%, 100.00%) and 98.81% (95% CI: 93.54%, 99.97%) for M. bovis infection of monkeys, 100.00% (95% CI: 86.82%, 100.00%) and 94.85% (95% CI: 91.22%, 97.03%) for M. tb infection of humans. Furthermore, this MMEC/AG-iELISA likely detects M. caprae infection in roe deer. Thus this method has a promising application in serological TB surveillance for multiple animal species thereby providing evidence for taking further action in TB control.