Abstract Conidiobolomycosis is a fungal disease that affects the upper respiratory tract of animal mainly sheep in tropical region. There are no vaccines or efficient treatments for this pathology and identifying immunoreactive proteins could create new opportunities for preventing or diagnosing this disease. The objectives of this study were to characterize the Conidiobolus lamprauges enolase-gene (eno) and evaluate its immunogenic capacity in naturally infected sheep serum. Gene amplification of eno gene from C. lamprauges resulted in a 1305 bp amplicon and 434 residues of deduced amino acid with a predicted molecular weight of 47.2 kDa and it had a signature enolase sequence. Anti-recombinant enolase IgG showed 80% of sensibility and 81.8% of specifity, with no cross-reactivity detected in sheep with pythiosis. Therefore, this study demonstrated that recombinant ENO from C. lamprauges is immunogenic against the sera of sheep that were naturally infected with conidiobolomycosis. The detection of specific anti-enolase antibodies in animals shows that the protein is a promising candidate antigen for the development of effective vaccines against the disease or diagnostic tests.