A case of fatal Mycobacterium tuberculosis infection was diagnosed postmortem in a captive 33-yr-old male black rhinoceros (Diceros bicornis) after a nonspecific illness in April 2013. Retrospective testing of sera from this individual revealed that it had been seroreactive by ElephantTB STAT-PAK, dual-path platform VetTB, and multi-antigen print immunoassay for over 12 yr prior to death. Although samples collected at the time of intradermal tuberculin test performed in October 2000 were nonreactive in all three serologic assays, the animal appeared to seroconvert approximately 2.5 wk after the skin test administration. The antibody response remained detectable for the duration of the animal's life (12 yr), indicating ongoing immunologic stimulation. The current case report supports the use of serologic assays for diagnosis of TB in black rhinoceros and may provide information for earlier detection. However, further research is needed to develop tools for recognition of mycobacterial infections in rhinoceros.