The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide（3DOM GTD） film was modified on the indium-tin oxide（ITO） electrode surface and cytochrome c（Cyt c） was successfully immobilized on the 3DOM GTD/ITO.A kind of Cyt c enzyme biosensor（Cyt c/3DOM GTD/ITO） was prepared.The properties of 3DOM GTD film were characterized by transmission electron microscopy（TEM） and scanning electron microscopy（SEM）.UV-Vis spectrum indicated that the Cyt c immobilized on the film retained its biological activity and the secondary structure of Cyt c was not destroyed.The direct electrochemistry（DET） of Cyt c on the surface of the modified electrode and its electric catalytic behavior to H2O2 were also studied.The results indicated that Cyt c showed an obviously direct electrochemistry response.The peak currents were linear with scan rates,which indicated that the electrochemical behavior of Cyt c/3DOM GTD/ITO electrode was a surface-controlled process.The Cyt c/3DOM GTD/ITO electrode showed a good catalytic performance to hydrogen peroxide,and a wide linear range of 3.0×10-7-1.7×10-5 mol/L was obtained with a detection limit（S/N=3） of 3.6×10-8 mol/L and a rapid response of 5 s.The electrode showed a good reproducibility and stability.