In this study, a recombinant chimeric antigen (CA) ELISA was validated as a single test for both human and dog leishmaniasis. Serum panels included 327 human and 339 canine IFAT-positive and 1113 human and 1078 canine IFAT-negative samples. CA-ELISA was carried out using the same serum dilution, and labelled protein A as secondary reagent. Test performances were calculated using ROC analysis. For the human panel, the test showed diagnostic accuracy (DA) 0.974, specificity (Sp) 97.12%, sensitivity (Se) 91.44%, and concordance (K) 0.88. The dog panel showed DA 0.998, Sp 99.54%, Se 98.54%, and K 0.98. The proposed method is the best recombinant antigen-based ELISA, and can be used as IFAT substitute for mass screening.