Layered double hydroxides (Zn2Cr(OH)6X-nH2O LDH) containing (3-sulfopropyl)ferrocene-carboxylate (FcPSO3) and 1,1′-bis(3-sulfopropyl) ferrocene-carboxylate (Fc(PSO3)2) as interlayer anions (X) have been prepared by the coprecipitation method and characterized by PXRD, FTIR, SEM and XPS. The electrochemical behavior of these hybrid materials has been evaluated by cyclic voltammetry. A new amperometric biosensor based on the immobilization of glucose oxidase in ZnCr-FcPSO3 hybrid material was presented, the intercalated anions playing the role of mediators that shuttle electrons between the FAD centers in the enzyme and the electrode surface. The performance of the resulting biosensor for glucose determination under anaerobic conditions was evaluated by chronoamperometry at 0.5 V. The sensitivity (65 mA M-1 crrT2) determined in the concentration range 10-25 mMis higher than sensitivities reported for other glucose biosensors based on LDH host matrices. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.