A novel triple-lines lateral-flow assay (LFA) with enhanced sensitivity for the detection of Leishmania infantum DNA from dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the use of gold nanoparticle tags (AuNPs) connected with polyclonal secondary antibodies which recognize anti-FITC ones. The polyclonal nature of the secondary antibodies allowed their multiple connections with primary ones, giving rise to the enhancement of the AuNP plasmonics signal. Furthermore, an endogenous control consisting in the amplified dog 18S rRNA gene was introduced so as to avoid false negatives. Using this strategy 0.038 spiked Leishmania parasites per DNA amplification reaction (1 parasite/100 mL of DNA sample) were detected, being this detection limit lower than the obtained with other techniques in addition to the advantage of being a universal and simple sensing alternative with interest to be extended to several other biosensing scenarios.