Bio-nanocomposite films based on chitosan and manganese oxide nanoflake have been fabricated via the layer-by-layer (LBL) self-assembly technique. UV–vis absorption spectra showed that the subsequent growth of the nanocomposite film was regular and highly reproducible from layer to layer. X-ray photoelectron spectroscopy (XPS) spectra confirmed the incorporation of chitosan and manganese oxide nanoflake into the films. Scanning electron microscopy (SEM) images revealed that the nanocomposite film had a continuous surface and a layered structure. A sensitive hydrogen peroxide (H2O2) amperometric sensor was fabricated with the chitosan–manganese oxide nanoflake nanocompoite film. The sensor showed a rapid and linear response to H2O2 over the range from 2.5 × 10− 6 to 1.05 × 10− 3 M, with a sensitivity of 0.038 A M− 1 cm− 2.