Abstract: Stable nano-Ag colloids were obtained by reducing AgNO3 in the presence of polyoxyethylene lauryl ether as reducing agent and stabilizing agent. The obtained nano-Ag colloids were mixed with hemoglobin (Hb) solutions and coated onto the glass carbon electrode. The entrapped Hb showed excellent direct electrochemical behaviors with a formal potential of -0.303 V versus SCE. Meanwhile, the entrapped Hb in nano-Ag colloids retained its native structure, reflected by UV-Vis spectroscopy. A hydrogen peroxide sensor was constructed, based on and the catalytic activity towards H2O2 of the modified glass carbon electrode, with apparent Michaelis-Menten constant (Kmapp) of 47 μmol·L-1, indicated that the electrode had a highly affinity to H2O2. On the optimum conditions, the linearity range of H2O2 was 1.0×10-7-3.0×10-4mol·L-1 with detection limit (3S/N) of 2.0×10-8mol·L-1.