An efficient and novel non-enzymatic glucose sensing platform composed of self-assembled crystalline NiTiO₃/NiO nanoparticles is reported in this article. The mixed oxide nanoparticles have been synthesized using a facile sol–gel method mediated by evaporation induced self-assembly (EISA) in non-aqueous media. After annealing at 673 K the final product obtained was well characterized using various analytical tools like powder X-ray diffraction (PXRD) and scanning and transmission electron microscopy (SEM and TEM), along with energy dispersive X-ray spectroscopy (EDS)and UV-visible diffuse reflectance spectroscopy (DRS) studies which reveal that the nanomaterial is composed of ca. 30–35 nm sized nanocrystals that have an ilmenite NiTiO₃ structural phase with some percentage of bunsenite NiO. The NiTiO₃/NiO nanoparticles exhibit an outstanding electro-catalytic activity towards glucose oxidation in 0.1 M NaOH alkaline media at an applied potential of +0.55 V (vs. Ag/AgCl electrode). The glucose sensing investigation of this Ni–Ti electrode indicates a very high sensitivity and a low limit of detection (LOD) of 1454 μA mM⁻¹ cm⁻² and 0.06 μM, respectively. The glucose level in human blood serum was also tested with our sample which produced a satisfactory result. Thus, the NiTiO₃/NiO electrode can be used as a highly effective, economical, stable and non-air sensitive platform for non-enzymatic glucose detection.