This research work reports a novel label-free DNA-based biosensor for highly sensitive detection of Haemophilus influenzae (H. influenzae) in urine samples. The detection was based on direct hybridization of DNA applying DPASV methods in the presence of [Fe(CN)۶]۳-/۴- for recognition of this pathogenic agent. To attain this objective, Ni/Cr layered double hydroxides (LDH) were synthesized and cast on a gold electrode. Furthermore, electrochemically addition of AuNPs
was performed on the gold electrode and the modified electrode was effectively prepared. Well-fabricated genosensing bio-assay represented high specificity, sensitivity, and good performance for the determination of the H. influenzae genome. Under optimal circumstances, LOD and LOQ were lower than in the other studies. The fabricated biosensing assay presented high selective ability of one, two, and three-base mismatched sequences. In addition, negative control of the genosensing assay for investigation of the selectivity was performed. Likewise, reproducibility and repeatability of the related bio-assay were investigated as well. It is to be noted that the organized genosensing bio-assay can be straightforwardly reused and regenerated to assess the hybridization process. Subsequently, the new approach is suitable for rapid and quantitative detection of H. influenzae and is a good candidate for further tests on clinical samples.