The gold standard for the diagnosis of sporotrichosis consists of the isolation and identification of the fungus from clinical exudates and/or biopsy specimens. However, new technologies offer several advantages over the traditional methods because they are noninvasive and more sensitive in the differential diagnosis of infectious diseases. In the present study, we performed a validation, impact evaluation, and analysis of the applicability of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of sporotrichosis in different groups of patients in comparison with the reference protocols for the evaluation of diagnostic tests for infectious diseases. We used 177 serum samples that were collected between 1998 and 2008 from patients in a geographic area related to the Rio de Janeiro outbreak of sporotrichosis. The ELISA had a low rate of cross-reactivity based on the reference values for diagnostic tests, and the analysis of the receiver operating characteristic curve revealed an area under the curve of 0.9154. Furthermore, higher accuracy was observed in the serodiagnosis of infections in special groups of patients such as pediatric and human immunodeficiency virus–positive patients. We concluded that this ELISA had a good clinical serological correlation and, therefore, can be considered a new diagnostic tool applicable to all clinical presentations of sporotrichosis.