Objectives: Accurate diagnosis of tuberculosis (TB) disease is a cornerstone in the management of disease spread and initiation of proper treatment. Detection of Mycobacterium tuberculosis (MTB) antigens in biological fluids might be a reliable way to directly diagnose TB disease. The aim of the current study is to investigate selected TB antigens as potential diagnostic serum biomarkers. Methods: We have explored the detection of three MTB antigens, namely Rv2324, Rv2435c and Rv1284, in serum specimens from confirmed pulmonary TB patients. In addition, serum specimens from Bacillus Calmette-Guérin (BCG)-vaccinated healthy adults and BCG-unvaccinated infants were also tested as negative controls. The three antigens were recombinantly expressed in Escherichia coli and purified using affinity chromatography. Murine antibodies against the three antigens were raised by mice immunization with corresponding recombinant antigens. TB antigen detection in the serum specimens was carried out by the dot blot test. Results: Serum samples from TB patients showed significant reactivity (94.3-100%) with the antigen-specific antibodies. Surprisingly, the three antigens were also detectable in serum specimens (93.1-100%) from healthy adults. On the contrary, the three antigens were barely detectable in sera from BCG-unvaccinated infants (0-14%). Conclusion: These finding, supported by recent literature, highlight the significant interference of BCG vaccination with the diagnosis of TB disease by direct antigen detection. Nevertheless, the three tested antigens could represent useful TB serum biomarkers in countries where BCG vaccination is not routinely performed.