In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.