Bovine tuberculosis is one of the most dangerous zoonotic diseases. Despite the near-complete elimination of the disease from cattle breeding in Poland achieved in 2009, its re-emergence is now observed. Globally, the number of human cases is underestimated and the importance of free-living animals as reservoirs of tuberculosis is growing. As a species highly susceptible to Mycobacterium tuberculosis complex infection, the European bison (Bison bonasus) has a role in the transmission of the disease in Poland. The purpose of the investigation was to assess the epidemiological situation of tuberculosis in Polish European bison serologically. A total of 460 serum samples were collected from 436 European bison from 15 out of 26 national populations between 2013 and 2020. An M. bovis ELISA was used, and its sensitivity and specificity were assessed with an eyelid tuberculin skin test (TST) and interferon gamma release assay (IGRA). Mycobacterium bovis antibodies were detected in nine serum samples. The presence of antibodies was found in two animals from the Białowieża Forest (1.2% of the population), and one each from the Borecka Forest (2.4%) and the Warsaw Zoo (14.3%). One European bison among the 14 sampled (7.1%) from Smardzewice was positive on five occasions. Other samples from Smardzewice and the Bieszczady Mountains, where tuberculosis had previously been reported, were negative. ELISA testing is an effective, easy and cost-efficient tool for monitoring of tuberculosis-naïve populations. Serological testing in tuberculosis control programmes can significantly improve the detection of infected herds. Antibody ELISAs may supplement TST and IGRA, but cannot replace them.