Sol-gel synthesized nickel oxide (NiO) film deposited onto indium tin oxide (ITO) coated glass plate has been utilized for the development of sensitive and stable DNA biosensor and demonstrated for diagnosis of visceral leishmaniasis also known as Kala-azar. Leishmania specific sensor is developed by immobilizing 23mer DNA sequence (oligonucleotide) identified from 18S rRNA gene sequences from Leishmania donovani. Characterization studies like X-Ray Diffraction and Scanning Electron Microscopy revealed the formation of nano-structured NiO, while immobilization of single strand (ss)-DNA of Leishmania was supported by UV-visible, Fourier Transform Infrared Spectroscopy and Scanning Electron Microscopy techniques. Response studies of ss-DNA/NiO/ITO bioelectrode are carried out using differential pulsed voltammetry in presence of methylene blue redox dye as a redox mediator. A linear response is obtained in the wide concentration range of 2 pg ml(-1) to 2 μg ml(-1) of complementary target genomic DNA (disease DNA) within the variation of 10% for 5 sets of studies. The observed results hold promise not only for diagnosis of Kala-azar patients but also hold enormous potential of the nano-NiO based probe for development of stable and sensitive biosensors.