Using layered double hydroxides (LDHs)-chitosan (Chit) composite as enzyme immobilization matrix, the developments of cholesterol biosensor were achieved by both electrooxidation and electroreduction of the enzymatic-generated hydrogen peroxide based on cholesterol oxidase (ChOx) and horseradish peroxidase (HRP). Amperometric detection of cholesterol was evaluated by holding the modified electrode at 0.55 V and 0 V (vs. SCE), for ChOx/LDHs-Chit/Pt and ChOx/HRP/LDHs-Chit/GCE, respectively. The linear dynamic ranges of anodic and cathodic detections of cholesterol were between 5 x 10(-7) to 8 x 10(-4) M and 4 x 10(-8) to 6 x 10(-4) M, respectively. The detection limits were determined to be of 1 x 10(-7) and 4 x 10(-8) M with anodic and cathodic processes, respectively. At potential of 0 V, ChOx/HRP/LDHs-Chit/GCE bienzymatic mediated system exhibited excellent selectivity and stability. (C) 2011 Elsevier B.V. All rights reserved.