Stable films of biopolymer chitosan and carbon nanotubes were prepared by a layer-by-layer self-assembly technique. Atomic force microscopy, scanning electron microscopy, cyclic voltammetry, and LTV-vis spectroscopy were used to characterize the film assembly. Atomic force microscopy and scanning electron microscopy showed that an even, stable film was formed. The UV-vis spectroscopy and cyclic voltammetry study indicated the uniform growth of the film. The property of the self-assembled multilayer film in promoting protein electron transfer was demonstrated by incorporating microperoxidase-11 in the film. Microperoxidase-11 in the multilayer film could transfer electrons with the electrode indicating that carbon nanotubes could wire the protein to the electrode. The electrocatalytic activity of the microperoxidase-11 containing multilayer film-modified electrode toward H2O2 and O-2 was investigated. The results showed that along with the increase in the assembled layers the electrocatalytic reduction potentials of H2O2 and O-2 shifted positively. The prepared multilayer film of chitosan and carbon nanotubes containing protein was a sensitive interface for electrocatalytic study.