An efficient and simple method has been demonstrated for the synthesis of nickel oxide nanostructures using urea as a capping agent. The nanosheet-like morphology was confirmed by scanning electron microscopy, crystalline nature was studied by using the X-ray diffraction (XRD) and surface area of nanomaterial was investigated by automated sorption analyzer. Then synthesized NiO nanostructures were used to fabricate the surface of glassy carbon electrode (GCE). The electrocatalytic parameters of modified NiO/GCE electrode were investigated by using various techniques such as electrochemical impedance spectroscopy (EIS), square wave voltammetry (SWV), differential pulse voltammetry (DPV), normal pulse voltammetry (NPV) and cyclic voltammetry (CV) and chronoamperometry. Various working experimental conditions were optimized in order to attain the highest sensitivity for the determination of urea and the highest peak current 1032 A of response were obtained at 100 M concentration of urea. A linear calibration plot was obtained for peak current versus concentration of urea in the range of 10 M urea to 80 M urea with a good detection limit of 2 M. The proposed working strategy was successfully employed for the estimation of urea in human urine samples and the obtained results are found satisfactory. The newly functional urea sensor can be exploited at large scale as an alternative analytical device beside to the other reported urea sensors.