Glassy carbon electrode was modified with different synthesized hybrid clay-based materials and tested in alkaline solution with and without H2O2. The hybrid materials were obtained by immobilizing hemoglobin (Hb) on acid activated (AA) clay, or on AA clay modified with different sodium dodecyl sul-fate (SDS) loadings. The obtained materials were characterized using DR UV vis and ESR spectroscopy, elemental analysis, and SEM. The characterization confirmed higher degree of hemoglobin incorpora-tion in the presence of SDS. The presence of SDS on the surface of clay particles resulted in the partial oxidation/denaturation of hemoglobin and formation of hemichrome. Cyclic voltammetry was used for the investigation of the electrochemical behavior of immobilized hemoglobin in alkaline solution. Two cathodic peaks at -0.45 V and -0.70 V were recorded and ascribed to the reduction of heme Fe(III)/Fe(II), and formation of HbFe(I) - highly reduced form of hemoglobin respectively. The latter peak reflects hemoglobin denaturation. The presence of H2O2 in the alkaline solution increased current intensities corresponding to both peaks (-0.45 V and -0.7 V). Linear response of peak current intensity vs. H2O2 concentration was monitored for all investigated samples within different H2O2 concentration ranges. The AA-SDSI.0-Hb electrode exhibited the highest current response with linear regression equation in the following form: I(mu A)= 7.99 +1.056 x1f1202] (mM) (R=0.996). The limit of detection of 28 mu M was estimated using the 3 sigma method. Different modified electrodes exhibited different degrees of denaturation resistance. The obtained val-ues of Michaelis-Menten constant indicated that prolonged cycling in the presence of SDS increases protein denaturation. (C) 2016 Elsevier B.V. All rights reserved.