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Transition metallic binary alloys have attracted enormous attention in regard to the non-enzymatic detection of glucose due to their high electrocatalytic activities induced by the synergistic effect between the individual metallic species. However, the easy aggregation of the bimetallic particles has limited their performance. Herein, a facile metal–organic framework (MOF)-derived strategy is developed to synthesize a hybrid containing binary Co–Ni nanoparticles decorated on an N-doped porous carbon matrix (Co_xNi_y/N-C) for the non-enzymatic detection of glucose. The Co/Ni ratio in the hybrid is investigated to regulate its electrocatalytic behaviors for glucose sensing. A hybrid with the optimal Co/Ni ratio of 1:1 displays two linear detection ranges (0.5 µM to 1 mM and 1 mM to 10 mM) with a detection limit of 0.11 µM for glucose. The feasibility of using this hybrid-modified SPE for glucose detection in real serum samples has also been validated.