Through electrostatic layer-by-layer (LBL) assembly, negatively charged citrate-stabilized platinum nanoparticles (PtNPs) and positively charged [tetrakis(N-methylpyridyl)porphyrinato] cobalt were alternately deposited on a 4-aminobenzoic acid-modified glassy carbon electrode and also on indium tin oxide substrates, directly forming the three-dimensional nanostructured materials. Thus-prepared multilayer films were characterized by UV-visible spectroscopy, surface plasmon resonance (SPR) spectroscopy, atomic force microscopy (AFM), and cyclic voltammetry. Regular growth of the multilayer films is monitored by UV-visible spectroscopy and SPR spectroscopy. AFM provides the morphology of the multilayer films. The PtNPs containing multilayer films exhibit high electrocatalytic activity for the reduction of dioxygen with high stability. Rotating disk electrode voltammetry and rotating ring-disk electrode voltammetry demonstrate that the PtNP-containing multilayer films can catalyze an almost four-electron reduction of O-2 to H2O in an air-saturated 0.5 M H2SO4 solution. Furthermore, the electrocatalytic activity of the films could be further tailored by simply choosing different cycles in the LBL process or more specifically the amount of the assembly components in the films. The high electrocatalytic activity and good stability for dioxygen reduction make the PtNP-containing multilayer films potential candidates for the efficient cathode material in fuel cells.