Direct electrochemistry of hemoglobin (Hb) is achieved by immobilizing Hb-chitosan on a gold colloid/3-aminopropyl triethylene silane/Prussian blue composite film-modified glassy carbon electrode. The modified electrode exhibits a pair of well defined and quasi-reversible peaks with a formal potential of 0.205 V. The immobilized Hb retains its biological activity and shows high catalytic activity to the reduction of hydrogen peroxide. Experimental conditions influencing the biosensor performances such as pH and potential are optimized and assessed. Under the optimized conditions, the catalytic currents are linear to the concentrations of H(2)O(2) in the ranges of 2-480 mu M. The detection limit is 0.1 mu M (S/N = 3). The electrochemical sensor has high stability and good reproducibility. Ultraviolet visible absorption spectra and Fourier transform infrared spectroscopy show that Hb keeps almost natural structure in the composite film. Therefore, the composite film is an alternative matrix for protein immobilization and biosensor preparation. (C) 2011 Elsevier B.V. All rights reserved.