In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.