Canine visceral leishmaniasis (CVL) is a major zoonosis that poses a growing challenge to public health services, as successful disease management requires sensitive, specific, and rapid diagnostic methods capable of identifying infected animals even at a subclinical level. The objective of this study was to evaluate the performance of the recombinant chimeric protein rMELEISH3 as an antigen in ELISA assays for the robust diagnosis of CVL. The protein was expressed in a bacterial system, purified by affinity chromatography, and evaluated through a series of serological assays using serum samples from dogs infected with Leishmania infantum. ROC curve analysis revealed a diagnostic sensitivity of 96.4%, a specificity of 100%, and an area under the curve of 0.996, indicating excellent discriminatory power. Furthermore, rMELEISH3 was recognized by antibodies present in the serum of dogs with low parasite loads, reinforcing the diagnostic potential of the assay in asymptomatic cases. It is concluded that the use of the recombinant antigen rMELEISH3 could significantly contribute to the improvement of CVL surveillance and control programs in endemic areas of Brazil and other countries, by offering a safe, reproducible and effective alternative to the methods currently recommended for the serological diagnosis of the disease.