As a new performance, nanostructured nickel oxide (Nano-NiO) thin film was considered at the optimized instrumental deposition conditions on the F-doped SnO2 conducting glass (FTO) by reactive radio frequency (RF) magnetron sputtering system. The attained uniform Nano-NiO thin film was successfully exploited to immobilize glucose oxidase enzyme (GOx) for impedimetric glucose quantification. Step by step film monitoring using field emission-scanning electron microscopy (FE-SEM) before and after enzyme immobilization established nanostructured NiO thin film with great isoelectric point (IEP similar to 10.7) was a suitable biosensing media for enzymes with low IEP (4.5). Observing of biosensor fabrication was accomplished using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The impedimetric results for invented FTO/Nano-NiO/GOx biosensor exhibited good sensitivity 4.45 k Omega/mM for glucose detection within 0.20-4.0 mM range, good electrocatalytic activity, and low detection limit as 24 mu M via appropriate enzyme activity and stability. The suggested disposable biosensor demonstrated a potential application for real sample analysis with unknown interferences.