We report on the synthesis of three-dimensionally ordered structure (3D) of macroporous cytochrome c (cyt-c) biofilms by using the inverted colloidal polystyrene crystal template technique and Triton X-100 as entrapping matrix. Electrochemical impedance spectroscopic (EIS) measurements reveal that the immobilized cyt-c molecules exhibit fast interfacial electron-communication rate. We found that the orientation of the cyt-c molecules entrapped in the 3D macroporous structure was determined by the interfacial electric field. Higher interfacial electric field limits the reorientational flexibility of the entrapped cyt-c molecules, resulting in lower intermolecular electron-communication rate constant (k degrees). Therefore, the highest intermolecular electron-communication rate constant can only be obtained at potentials approaching to the potential of zero charge of the gold electrode, since k degrees is mainly determined by the molecular orientation in the biofilm. In addition, the prepared biofilms with enhanced functional density could find potential application in the bioelectronic sensing system.