A novel composite film comprising cationic gemini surfactant butyl-alpha,omega-bis(dimethylcetylammonium bromide) (C16H33N(CH3)(2)-C4H8-N(CH3)(2)C16H33, C-16-C-4-C-16) and ionic liquid 1-octyl-3-methylimidazolium hexafluorophate (OMIMPF6) has been prepared. The composite film shows good biocompatibility and it can promote the direct electron transfer between hemoglobin (Hb) and glassy carbon (GC) electrode. On the C-16-C-4-C-16 (dissolved in ethanol)-OMIMPF6 film coated GC electrode, the immobilized Hb can exhibit a pair of well-defined, quasi -reversible and stable redox peaks with a formal potential of -0.317 V (vs SCE) in 0.10 M pH 7 phosphate buffer solutions. The electron transfer coefficient (alpha) of Hb is calculated to be 0.44 and the heterogeneous electron transfer rate constant is 6.08 s(-1). With the length of alkyl chains of gemini surfactant increasing and the ethanol concentration rising, the redox peaks of the resulting electrode C-16-C-4-C-16-OMIMPF6-Hb/GC become bigger. The electrode presents good electrocatalytic response to peroxide hydrogen. The kinetic parameters I-max and k(m) for the catalytic reaction are estimated. In addition, UV-vis spectra and reflectance absorption infrared spectra demonstrate that the Hb immobilized in the C-16-C-4-C-16-OMIMPF6 film almost retains the structure of native Hb. (c) 2008 Elsevier Ltd. All rights reserved.