In this work, a low cost glucose and methanol nonenzymatic sensor was prepared using nickel oxide (NiO) nanofilm electrodeposited on a bare Cu electrode. Electrochemical deposition was assisted with cetyl trimethylammonium bromide (CTAB) as a template. Scanning electron microscopy (SEM) was applied to observe the surface morphology of the modified electrode. Cyclic voltammetry (CV) and amperometry techniques were used to study the electrocatalytic behavior of NiO porous film in glucose and methanol detection. For glucose sensing, the electrode showed a linear relationship in the concentration range of 0.01-2.14 mM with a low limit of detection (LOD) 1.7 µM (signal/noise ratio (S/N)=3). Moreover, high sensitivities of 4.02 mA mM?1 cm?2 and 0.38 mA mM?1 cm?2 respectively in glucose and methanol monitoring suggested the modified electrode as an excellent sensor. The NiO-Cu modified electrode was relatively insensitive to common biological interferers. This sensor possessed good poison resistance towards chloride ions, and long term stability and significant selectivity towards glucose and methanol. Finally the proposed sensor was successfully applied for determination of glucose in human blood serum samples.