A novel hydrogen peroxide (H2O2) biosensor was developed by immobilizing hemoglobin on the gold colloid modified electrochemical pretreated glassy carbon electrode (PGCE) via the bridging of an ethylenediamine monolayer. This biosensor was characterized by UV-vis reflection spectroscopy (UV-vis), electrochemical impendence spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized Hb exhibited excellent electrocatalytic activity for hydrogen peroxide. The Michaelis-Menten constant (K (m)) was 3.6 mM. The currents were proportional to the H2O2 concentration from 2.6 x 10(-7) to 7.0 x 10(-3) M, and the detection limit was as low as 1.0 x 10(-7) M (S/N = 3).