Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazil, (Project: APQ 03097 15).In this work, a dual detection system based on an impedimetric immunosensor was developed for the lust time for the simultaneous detection of anti-Tgpanosoma cruzi and anti-Leishmania infanturn antibodies in human and dog serum samples. The IBMP 8.1 and rLcilA/rLci2B recombinant antigens were inunobilized over the surface of dual screen-printed carbon electrodes (W1 and W2) modified with poly (4-hydroxyphenylacetic acid). Under optimized conditions, the immunosensor recognized specific interactions for anti-T. cruzi antibodies up to a dilution of 1:10,240 and for anti-L. infant-tun up to 1:5120 in canine seams samples. Relative standard deviation (RSD) values of 2.8% for W1 and 3.6% for W2 were obtained for T. cruzi (W1) and L infannun antigen (W2) samples in three different electrodes for 3 days (n = 9). The immunosensor was stored at 4 °C for 8 weeks, with activity retention of 70.2% in W1 and 78.2% in W2. The results using the recombinant proteins revealed that all antigens discriminated between negative and positive samples (p < 0.0001) in both dog and human groups, as well as no cross-reactivity could be detected among sera with other infections. With this approach, immunosensor-based diagnostic tests achieved 100% accuracy, suggesting that the antigens are eligible to enter Phase-II studies.