The present study has been focused on a facile strategy to fabricate an electrochemical sensor based on nickel oxide decorated Fe3O4 nanoparticles/poly(p-aminohippuric acid)-sodium dodecyl sulfate nanocomposite immobilized on the modified glassy carbon electrode (NiO@ Fe3O4-SH/PPAH-SDS/GCE). PPAH-SDS film was prepared by electropolymerization and nickel oxide nanostructures were deposited on the Fe3O4-SH/PPAH-SDS by electrochemical method. The as-synthesized nanocomposite was characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) for morphological and elemental analysis, respectively and the electrochemical characteristics were studied by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods. The differential pulse voltammetry (DPV) was used for the sensitive determination of glucose in alkaline solution in two linear concentration ranges, 0.1-10.0 mu M and 10.0-300.0 mu M, with a detection limit of 0.13 mu M (S/N = 3) based on the first linear range. Meanwhile, the presented sensor showed a promising performance for the quantification of glucose in human serum as a real sample reflecting the practicability of fabricated glucose sensor.