Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis and pneumonia. However accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. An immunogenic protein of 226 kDa was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in E. coli as GST fusion proteins and recombinant products from the amino terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG ELISAs. The IgM ELISA was able to detect M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA was able to detect M. bovis-specific IgG three weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA had potential for use as a sensitive and specific assay to detect infection in cattle.