A modified electrode with conducting polymer (Polyaniline) and NiO nanoflowers was prepared to detect nitrite ions in drinking water. A simple method was used to prepare the NiO nanoflower (NiOnF). Several techniques characterized the as-prepared NiOnF to determine the chemical structure and surface morphology of the NiO, such as XRD, XPS, FT-IR, and TGA. The activity of the electrode toward nitrite sensing was investigated over a wide range of pH (i.e., 2 to 10). The amperometry method was used to determine the linear detection range and limit. Accordingly, the modified electrode GC/PANI/NiOnf showed a linear range of detection at 0.1–1 µM and 1–500 µM. At the same time, the limit of detection (LOD) was 9.7 and 64 nM for low and high concentrations, respectively. Furthermore, the kinetic characteristics of nitrite, such as diffusion and transport coefficients, were investigated in various media. Moreover, the charge transfer resistance was utilized for nitrite electrooxidation in different pH values by the electrochemical impedance technique (EIS). The anti-interfering criteria of the modified surfaces were utilized in the existence of many interfering cations in water (e.g., K+, Na+, Cu2+, Zn2+, Ba2+, Ca2+, Cr2+, Cd2+, Pd2+). A real sample of the Nile River was spiked with nitrite to study the activity of the electrode in a real case sample (response time ~4 s). The interaction between nitrite ions and NiO{100} surface was studied using DFT calculations as a function of adsorption energy.