In this paper we report the development of a sensitive and specific assay for the detection of tuberculosis (TB) in European badgers (Meles meles), based on the stimulation of lymphocytes in whole-blood culture and the subsequent detection of gamma-interferon (IFNγ) by sandwich ELISA. The comparative levels of IFNγ produced to bovine and avian tuberculin (B–A) was used as the basis of determining the TB status of badgers, resulting in a more sensitive test than that based on the defined Mycobacterium bovis antigens ESAT6 and CFP10. The assay was evaluated using 235 badgers. The IFNγ EIA (enzyme immunoassay) based on a monoclonal pair (mEIA) was more sensitive than one using a rabbit polyclonal antiserum (pEIA). At a specificity of 93.6%, the mEIA was 80.9% sensitive, compared to a sensitivity of 74.5% for the pEIA. At the same specificity as the EIA, the current serological ELISA test for TB in badgers (Brock test) had a sensitivity of 48.9%. Only one of the culture positive badgers missed by the mEIA was correctly diagnosed by the Brock test, suggesting that the combination of both a T-cell and serological test has little diagnostic advantage.