This study described fabrication, characterization, and application of multilayer films based on layer-by-layer assembly of ferrocene poly(ethylenimine) and gold nanoparticles. Assembly process of the multilayer film was investigated by atomic force microscopy, UV-visible absorption spectroscopy and electrochemical impedance spectroscopy. The multilayer films exhibited a pair of well-defined redox peaks as revealed by cyclic voltammetry, as well as bifunctional and fine-tunable electrocatalysis for oxidation of ascorbic acid and reduction of oxygen. Both the outer layer and layer number had effect on the electrocatalytic response. Electrocatalytic activity of the films could be controlled with assemblies at the nanoscale level by simply adjusting deposition cycles or amount of component in the films.