Indium-tin oxide (ITO) surfaces have become a very important material for use as a conducting substrate, recently. Immobilization of biological molecule onto the ITO is a fundamental step in the development of biosensors. In this paper, attachment of albumin was carried out using ITO as a support. Albumin was covalently immobilized onto self-assembled monolayer (SAM) of (3-isocyanatopropyl) triethoxysilane (IPTES) on ITO surface. Isocyanato-functionalized organic film was prepared by self-assembling on active ITO surface. After that, human serum albumin (HAS) which was prepared in Phosphate-Buffered Saline Solution (PBS: pH 7.4, 0.1 M), was covalently immobilized on IPTES modified ITO surface. The study here can help us understand the influence of monolayer and electrochemical behavior on ITO surface. Characterization of stepwise changes of active ITO, IPTES/ITO and albumin/IPTES/ITO surfaces were carried out using contact angle (CA) measurements, fluorescence microscopy, atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. © 2009 Elsevier B.V. All rights reserved.