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A novel matrix, multiwalled carbon nanotubes supported nickel oxide nanoparticles composite nanomaterial (NiO@MWNTs), for immobilization of protein and biosensing was designed using a simple and effective hydrothermal method. Using myoglobin (Mb) as a model, the direct electrochemistry of immobilized Mb indicated the matrix could accelerate the electron transfer between protein's active sites and the electrode. The modified electrode shows excellent electrocatalytic activity toward the reduction of H 2 O 2 without the help of an electron mediator. The simple operation, fast response, acceptable stability, and reproducibility of the proposed biosensor indicated its promising application in protein immobilization and preparation of the third generation biosensors.