This research introduces a non-enzymatic electrochemical sensor utilizing flower-like nickel ox-ide/carbon (fl-NiO/C) microspheres for the precise detection of L-glutamic acid (LGA), a crucial neurotransmitter in the field of healthcare and a frequently utilized food additive and flavor en-hancer. The fl-NiO/C were synthesized with controllable microstructures using metal-organic frameworks (MOFs) as precursors followed by a simple calcination process. The uniformly synthe-sized fl-NiO/C microspheres were further characterized using Fourier Transform Infrared Spec-troscopy (FTIR), X-ray powder diffraction (XRD), Energy-dispersive X-ray spectroscopy (EDX), and Field Emission Scanning Electron Microscopy (FE-SEM). The fl-NiO/C was utilized as a modi-fier on the surface of a glassy carbon electrode, and an impedimetric sensor based on Electro-chemical Impedance Spectroscopy (EIS) was developed for the detection of LGA. The proposed sensor demonstrated excellent catalytic activity and selectivity towards LGA across a broad con-centration range of 10-800 μM with a sensitivity of 486.9 µA.mM-1.cm-2 and a detection limit of 1.28 µM (S/N=3). The sensor was also employed to identify LGA in blood plasma samples, yield-ing results that align with those obtained through HPLC. This achievement highlights the poten-tial of fl-NiO/C microspheres in advancing cutting-edge biosensing applications.