A novel biopolymer/room-temperature ionic liquid composite film based on carrageenan, room temperature ionic liquid (IL) [1-butyl-3-methylimidazolium tetra?uoroborate ([BMIM]BF4)] was explored for immobilization of hemoglobin (Hb) and construction of biosensor. Direct electrochemistry and electrocatalytic behaviors of Hb entrapped in the IL-carrageenan composite ?lm on the surface of glassy carbon electrode (GCE) were investigated. UV-vis spectroscopy demonstrated that Hb in the IL-carrageenan composite ?lm could retain its native secondary structure. A pair of well-de?ned redox peaks of Hb was obtained at the Hb-IL-carrageenan composite ?lm modi?ed electrode through direct electron transfer between the protein and the underlying electrode. The heterogeneous electron transfer rate constant (ks) was 2.02 s−1, indicating great facilitation of the electron transfer between Hb and IL-carrageenan composite film modi?ed electrode. The modi?ed electrode showed excellent electrocatalytic activity toward reduction of hydrogen peroxide with a linear range of 5.0×10−6 to 1.5×10−4 mol/L and the detection limit was 2.12×10−7 mol/L (S/N=3). The apparent Michaelis-Menten constant KMapp for hydrogen peroxide was estimated to be 0.02 mmol/L, indicating that the biosensor possessed high af?nity to hydrogen peroxide. In addition, the proposed biosensor showed good reproducibility and stability.