Layered nanocomposite of methylene blue (MB)-intercalated vanadium oxide was obtained through a simple hydrothermal synthesis method using MB, V2O5, and NaI as starting materials. The intercalation reaction was proven to be successful using X-ray diffraction pattern. The MB-V2O5 nanocomposite was characterized using a scanning electron micrograph, infrared spectra, thermogravimetric analysis, UV spectra, and electrochemical measurements. The intercalated MB cations showed a fine diffusion-controlled electrochemical redox process and facilitated the immobilized horseradish peroxidase's (HRP) good catalytic reduction upon H2O2. The as-prepared MB-V2O5/HRP biosensor showed a linear response to H2O2 over a range from 2.0 x 10(-6) to 9.5 x 10(-5) M with a detection limit of 9.7 x 10(-7) M (S/N ratio = 3).