Myoglobin/multi-walled carbon nanotubes/chitosan film (Mb/MWNTs/Cs) electrodes were fabricated based on cross-linking myoglobin (Mb) with multi-walled carbon nanotubes/chitosan (MWNTs/Cs) composite film coated on a glassy carbon electrode (GCE). Direct electrochemistry and electrocatalysis of Mb on the Mb/MWNTs/Cs/GCE were studied. The Mb/MWNTs/Cs/GCE exhibited excellent electrocatalytic activity and rapid response for H2O2 in the absence of a mediator. The linear range of detection towards H2O2 was from 3.79×10 to 5.50×10 mol L by the method of amperometric i-t curve. The Mb/MWNTs/Cs/GCE had good repeatability and stability for the determination of H2O2.