A graphene (GR) and Mg2Al layered double hydroxide (LDH) composite was prepared and used for the immobilization of hemoglobin (Hb) on a carbon ionic liquid electrode (CILE) to obtain an electrochemical biosensor. Morphology of the materials was analyzed by scanning electron microscopy and transmission electron microscopy. Spectroscopic experiments were performed to characterize the conformational changes of Hb in the GR-LDH membrane. Electrochemical investigation showed a pair of well-defined quasi-reversible redox peaks appeared with redox peak potentials as -0.145 V (E-pa) and -0.248 V (E-pc) in phosphate buffer solution (0.1 mol L-1, pH 3.0) at the scan rate of 100 mV s(-1), which indicated that direct electrochemistry of Hb was realized on the modified electrode. So the presence of GR-LDH composite could facilitate the electron transfer rate between CILE and the electroactive center of Hb. The immobilized Hb in GR-LDH modified electrode exhibited excellent electrocatalytic reduction to trichloroacetic acid in the concentration range from 1.6 to 25.0 mmol L-1 with the detection limit as 0.534 mmol L-1 by cyclic voltammetry, which indicated the successfully preparation of a third-generation electrochemical biosensor. (C) 2012 Elsevier Ltd. All rights reserved.