Abstract A robust and effective composite film combined the benefits of room temperature ionic liquid (RTIL), chitosan (Chi) and multi-wall carbon nanotubes (MWNTs) was prepared. Cytochrome c (Cyt c) was successfully immobilized on glassy carbon electrode (GCE) surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Cyt c were investigated in detail. A pair of well-defined and quasi-reversible redox peaks of Cyt c was obtained in 0.1 mol L −1 pH 7.0 phosphate buffer solution (PBS), indicating the Chi–RTIL–MWNTs film showed an obvious promotion for the direct electron transfer between Cyt c and the underlying electrode. The immobilized Cyt c exhibited an excellent electrocatalytic activity towards the reduction of H 2O 2. The catalysis current was linear to H 2O 2 concentration in the range of 2.0 × 10 −6 to 2.6 × 10 −4 mol L −1, with a detection limit of 8.0 × 10 −7 mol L −1 (S/N = 3). The apparent Michaelis–Menten constant ( K m) was calculated to be 0.45 ± 0.02 mmol L −1. Moreover, the modified electrode displayed a rapid response (5 s) to H 2O 2, and possessed good stability and reproducibility. Based on the composite film, a third-generation reagentless biosensor could be constructed for the determination of H 2O 2.