The application of nanolayered inorganic materials as supports for immobilizing biomolecules has attracted attention because the host structures not only have "flexible pores" to adapt to the size of the biomolecule guests but they also protect the immobilized biomolecules from disturbances created by environmental variations. In this paper, layered titanate sheets (TNSs) were used as support matrixes for immobilizing myoglobin (Mb) to fabricate protein electrodes suitable for studying the direct electron transfer between the redox centers of proteins and the electrode and establish an example of a mediator-free (third-generation) biosensor. The uniform., porous morphology and ordered structure of the protein electrode were characterized by scanning electron microscopy, high-resolution transmission electron microscopy, and X-ray diffraction. The native structure of the intercalated Mb was maintained in the TNSs, and its behavior was characterized by UV-vis and Fourier-transform IR spectroscopy. Immobilized Mb in TNS films showed a fast direct electron transfer for the Mb-Fe-III/Fe-II redox couple. Based on the direct electron transfer of the immobilized Mb, the protein electrode exhibited excellent catalytic performance for H2O2. Furthermore, in order to improve the stability of immobilized Mb in the TNS films, the thermal stability of the protein electrode and its electrochemical catalytic reduction of H2O2 in a low pH environment were discussed.