Mycobacterium farcinogenes is the causal agent of bovine farcy, a chronic infectious disease of zebu cattle in some parts of tropical Africa. Whole cell homologous antigen of M. farcinogenes was used in the standardization and evaluation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against bovine farcy using sera from confirmed bovine farcy and from bovine farcy-free cattle. The cut-off optical density (OD) value was decided at 1.8 using filter 405 nm after one hour of incubation at 37 degrees C. Accordingly, 115 out of 124 (92.7%) serum samples from clinically proven bovine farcy cattle were reported sero-positive. Sera from cattle infected with M. avium and M. paratuberculosis revealed OD value <1.8, indicating the differential diagnostic ability of M. farcinogenes antigen. Our test sensitivity was 92.7% and specificity was 97%, therefore could be routinely employed to support early clinical diagnosis, epidemiological surveys and for screening animals before exportation to farcy-free regions.