A novel nickel ion implanted-modified indium tin oxide (Ni/ITO) electrode was prepared, and the existence of nickel nanoparticles was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Upon fabrication of Ni/ITO with hemoglobin (Hb) and Nafion, Nafion/Hb/Ni/ITO for hydrogen peroxide (H2O2) biosensor was developed. Ultraviolet-visible (UV-vis) spectroscopy shows that Hb remains in its native structure. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and amperometry were employed for electrochemical studies. The results show that the presence of nickel nanoparticles can enhance the electrochemical response of Hb. A pair of well-defined and reversible CV peaks for the protein heme Fe(III)/Fe(II) redox couples was obtained at the prepared electrode Nafion/Hb/Ni/ITO. The biosensor exhibits good electrocatalytic activity toward H2O2. Amperometric response varies linearly with H2O2 concentration ranging from 0.8 mu M to 122 mu M with a detection limit of 0.5 mu M. The apparent Michaelis-Menten constant is calculated to be 0.325 mM indicating a high affinity to H2O2. Moreover, the modified biosensor exhibits high sensibility, good reproducibility, and long-term stability. (C) 2013 The Electrochemical Society. All rights reserved.