A novel composite film based on platinum nanoparticles (PtNPs), multi-walled carbon nanotubes (MWCNTs), room temperature ionic liquid (RTIL) and Nafion, is an effective matrix to immobilize myoglobin (Mb). Platinum nanoparticles were deposited on MWCNTs and characterized by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) techniques. Direct electrochemistry and electrocatalysis of Mb were studied in detail using differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) methods. Using CA, the mediator-free biosensor response was linear with hydrogen peroxide (H2O2) concentration in the range of 0.5 to 210 M with a detection limit of 0.1 μM.