In this paper, nanoporous titanium dioxide (TiO2) film was used for cytochrome c (cyt c) immobilization as an electrode substrate for electrochemical redox activity of the adsorbed cyt c. The result of cyclic voltammetry exhibited a pair of well-defined and quasi-reversible peaks for direct electron transfer of cyt c (formal potential [E-0' = (E-pa + E-pc)/2] of 53 mV versus Ag/AgCl). In addition the effect of metal and nonmetal ions (Au, S) co-doping on the efficiency of TiO2 nanoparticles (prepared by combining sol-gel and photo-deposition methods) on the cyt c immobilization process was investigated. The results exhibited that the Au, S-co-doped TiO2 (Au/S-TiO2) with a spheroidal shape demonstrates a smaller grain size than the pure TiO2. Meanwhile, the UV-vis DRS of Au/S-TiO2 showed a considerable red shift to the visible region. As a result, it was found that 4% Au/0.1% S-TiO2 had the highest efficiency for cytochrome c immobilization. The results showed that the peak currents were higher after the annealing of the TiO2 film. This observation suggests that the use of TiO2 films may be advantageous for the development of nanoporous biosensors employing reductive electrochemistry. (C) 2015 Elsevier B.V. All rights reserved.