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A NiFe alloy nanoparticle/graphene oxide hybrid (NiFe/GO) was prepared for electrochemical glucose sensing. The as-prepared NiFe/GO hybrid was characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The results indicated that NiFe alloy nanoparticles can be successfully deposited on GO. The electrochemical glucose sensing performance of the as-prepared NiFe/GO hybrid was studied by cyclic voltammetry and amperometric measurement. Results showed that the NiFe/GO-modified glassy carbon electrode had sensitivity of 173 μA mM^−1 cm^−2 for glucose sensing with a linear range up to 5 mM, which is superior to that of commonly used Ni nanoparticles. Furthermore, high selectivity for glucose detection could be achieved by the NiFe/GO hybrid. All the results demonstrated that the NiFe/GO hybrid has promise for application in electrochemical glucose sensing.