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It is estimated that there are 400000 new cases of visceral leishmaniasis each year, with about 30,000 deaths. Therefore, detection of this pathogen and its species is highly vital for overall health of the community. In the present research, a DNA-based biosensor, namely genosensor, was introduced for detection of genomic DNA of Leishmania infantum. The genosensor was fabricated based on the transduction of cadmium sulfide nanosheets and recognition of a particular single stranded DNA sequence, and worked in label-, marker-, tag- and PCR-free manners. Impedimetric measurements were performed in a wide range of frequency (recording Nyquist diagrams) without applying external force (working at open circuit potential) upon hybridization of DNA targets with the cadmium sulfide nanosheets surface-immobilized probe. The genosensor detected the complementary DNA strand in a concentration range of 1.0 × 10−14 to 1.0 × 10−6 mol L−1 and a detection limit (DL) of 0.81 fmol L−1 (6.5 fg mL−1), and genomic DNA of Leishmania infantum in a concentration range of 5–50 ng μL−1 and a DL of 1.2 ng μL−1. The genosensor had a very good selectivity, fabrication reproducibility and stability, and was applicable for practical applications. Image 1 • A specific DNA sequence was selected from gene sequence of Leishmania infantum. • The DNA sequence was immobilized on cadmium sulfide nanosheets to fabricate a genosensor. • The genosensor detected targets with a nice selectivity. • Leishmania infantum genomic DNA was quantified by the genosensor in clinical samples. [ABSTRACT FROM AUTHOR]