The laboratory diagnosis of visceral leishmaniasis (VL) presents limitations related to its sensibility and/or specificity. In this context, the aim of this study was to evaluate an enzyme-linked immunoassay to detect IgG antibodies against Leishmania infantum exo-antigens for diagnosis of VL, called ELISA-Exo. This assay was applied in 309 masked serum samples from VL, tegumentary leishmaniasis, Chagas disease, schistosomiasis mansoni, malaria patients, and healthy individuals. The results were compared with those from ELISA using rK39 as antigen (ELISA-rK39). The ELISA assays presented sensitivity of 96.8% and 98.4% (p = 0.68), specificity of 92.4% for both, and diagnostic accuracy of 94.2% and 94.8% (p = 0.48) by the ELISA-Exo and ELISA-rK39, respectively. An excellent agreement beyond chance (Kappa index = 0.82) was obtained when the results from ELISA assays were cross-tabulated. The Western blotting showed that false-positive results presented by ELISA-Exo probably were produced by cross-reactivity of antigens shared with the species of the family Trypanosomatidae. In the future, an immunoproteomic approach can contribute for identification of main immunoreactive L. infantum exo-antigens.