A novel polymer/room-temperature ionic liquid (RTIL) composite material based on chitosan (Chi) and 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMIM center dot BF4) was explored. The composite system can be readily used as an immobilization matrix to entrap proteins and enzymes. Hemoglobin (Hb) was chosen as a model protein to investigate the composite system. A pair of well-defined quasireversible redox peaks of hemoglobin were obtained at the Chi-BMIM center dot BF4-Hb composite-film-modified glassy carbon (GC) electrode by direct electron transfer between the protein and the GC electrode. Dramatically enhanced biocatalytic activity was exemplified at the Chi-BMIM center dot BF4-Rb/GC electrode by the reduction of oxygen and trichloroacetic acid. Thermogravimetric analysis (TGA) suggests that the Chi-BMIM center dot BF4-Hb composite has higher thermal stability than Chi-Hb itself. The Chi-BMIM center dot BF4-Hb film was also characterized by UV-visible spectra, indicating excellent stability in solution and good biocompatibility for protein. The unique composite material based on polymer and ionic liquid can find wide potential applications in direct electrochemistry, biosensors, and biocatalysis.