Nickel-based metal−organic frameworks, denoted as three-dimensional nickel trimesic acid frameworks (3D Ni-TMAF), are gaining significant attention for their application in nonenzymatic glucose sensing due to their unique properties. Ni-MOFs possess a high surface area, tunable pore structures, and excellent electrochemical activity, which makes them ideal for facilitating electron transfer and enhancing the catalytic oxidation of glucose. This research describes a new electrochemical enzyme-mimic glucose biosensor in biological solutions that utilizes 3D nanospheres Ni-TMAF created layer-by-layer on a highly porous nickel substrate. The Ni-TMAF based on the nonenzymatic electrochemical glucose oxidation represent the promising approach, leveraging the unique properties of Ni-TMAF to provide efficient, stable, and potentially more cost-effective alternatives to traditional enzyme-mimic sensors. The MOF is synthesized from trimesic acid (TMA) and nickel nitrate hexahydrate through a solvothermal reaction process. The resulting Ni-TMAF utilizes the three-dimensional nanospheres of crystalline porous structure with a large surface area and numerous active sites for catalytic reaction toward glucose. Ni-TMAF are indeed known for their excellent electrocatalytic activity, particularly in the context of glucose oxidation under alkaline conditions. The nickel centers in the Ni-TMAF facilitate efficient electron transfer and redox reactions, leading to the high sensitivity of 203.89 μA μM −1 cm −2 and lower LOD of 0.33 μM and fast response time of <3 s in glucose sensors. Their stability, cost-effectiveness, and high performance make 3D Ni-TMAF a promising material for nonenzymatic electrochemical glucose sensors.