A novel triple lines lateral-flow assay (LFA) with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the gold nanoparticle tags (AuNPs) conjugated to polyclonal secondary antibodies, which recognize anti-FITC antibodies. The polyclonal nature of the secondary antibodies allows for multiple binding to primary antibodies, leading to enhanced AuNP plasmonics signal. Furthermore, endogenous control consisting of the amplified dog 18S rRNA gene was introduced to avoid false negatives. Using this strategy, 0.038 spiked Leishmania parasites per DNA amplification reaction (1 parasite/100 μL of DNA sample) were detected. Detection limit of LFA was found to be lower than that of the conventional techniques. In summary, our novel LFA design is a universal and simple sensing alternative that can be extended to several other biosensing scenarios. We acknowledge the E.U.’s support under FP7-SME2012-1 contract number 315653 “POC4PETS”. ICN2 also acknowledges support of the Spanish MINECO under Project MAT2011–25870 and through the Severo Ochoa Centers of Excellence Program under Grant SEV-2013-0295. Peer reviewed