A layer-by-layer route to prepare nanoporous Au film materials on transparent ITO substrates is reported by alternatively assembling Au and Ag nanoparticles through 1,5-pentanedithiol as a cross-linker, followed by that Ag nanoparticles are dissolved at room temperature in HAuCl4 solution. Electron transfer of cytochrome c (cyt. c) - an excellent model for investigation of biomolecules, is greatly facilitated at the nanoporous Au film with electron transfer rate constant (ks) of 3.9s(-1). Meanwhile, cyt. c adsorped onto the nanoporous Au film still maintain its enzymatic activity toward H2O2. On the basis of these experimental results, the cyt. c-nanoporous Au film is exploited to an amperometric biosensor for H2O2 with high selectivity, broad linear range, low detection limit, and long stability.