A simple strategy to fabricate porous three-dimensional gold-film electrodes using gold-coated microspheres (AuMS) is presented. The AuMS deposited on gold (AuMS/Au) are electrically interconnected, and show unique electrochemical properties indicating porous surface structures. The AuMS/Au electrodes exhibit electrocatalytic activities for oxygen reduction and peroxide oxidation, which indicates that the apparent macroporous structures also retain porous regions with nanometer-scale dimensions. The porous structures on AuMS/Au electrodes also provide a selective voltammetric response for glucose against ascorbic acid due to the different electron-transfer kinetics. Since AuMS/Au electrodes are simple to prepare compared with previously suggested systems, the present system could offer potential in electroanalytical applications as new gold-based substrates for further modifications of surfaces.