Display omitted •Innovative peptides SPR imunossensor for canine leishmaniasis diagnosis.•Comparison of two immunogenic synthetic peptides as bioreceptors.•This simple label-free methodology allowed fast, sensitive, and selective analysis.•Via PCA was able to observe that even smaller times of detection can be explored.•Excellent ability to discriminate between positive and negative canine real samples. A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.