Cases of feline tuberculosis (TB) can be challenging to diagnose. Currently, this is achieved through a combination of mycobacterial culture, polymerase chain reaction (PCR), or interferon-gamma release assay (IGRA); however, these each have limitations. There is limited data regarding the use of humoral immunodiagnostics for TB in cats. Therefore, we sought to develop an enzyme-linked immunosorbent assay (ELISA) to further facilitate the diagnosis of feline TB. A comparative PPD (purified protein derivative) antibody ELISA was optimised for use on serum and plasma, and was tested against samples from 14 cats with culture-confirmed TB and 24 uninfected controls. Selection of an appropriate positive cut-off value based on receiver-operator characteristic curve analysis gave test sensitivity of 64.3 % and specificity of 100 %. When tested on further samples from cats with strongly suspected mycobacteriosis, 32.9 % (23/70) were antibody positive. Notably, positive results were recorded in cats that failed to respond to the IGRA, and in one PCR and IGRA negative cat. No positive responses were identified in cats with non-tuberculous mycobacterial infections, or with non-mycobacterial diseases (n = 12). Therefore, antibody-based diagnostics may be useful adjunctive tests for cases of TB missed by the IGRA, helping protect both feline and, in turn, human health.