In this study, a Sphingomonas Strain 264 isolated from pulp and paper biofilms was used for electrochemical studies towards applications in biosensor development based on its high ability to produce extracellular polymeric substances (EPS), along with its rapid growth and tendency to flocculate. The Strain 264 biofilm was directly grown on an An thin film and was studied with a combination of optical and electrochemical methods. The Strain 264 biofilm is stable in a wide potential range from -0.7 V to 1.0 V versus SCE; it is partially oxidized when the potential is above 1.0 V. In addition, the Strain 264 biofilm was used as an immobilization matrix for biosensor fabrication using horseradish peroxidase (HRP) as a model enzyme. Our electrochemical measurements show that the fabricated biosensor based on immobilized HRP in the Strain 264 biofilm exhibits a wide linear range (1-18 mM) and a high sensitivity (90 mu A mM(-1)) for H2O2 detection, demonstrating that the formed biofilm provides a favourable microenvironment for enzyme to retain its activity. This study has also shown that direct growth of biofilms is a feasible and effective approach for promising applications of biofilms in electrochemical biosensor development. (C) 2007 Elsevier B.V. All rights reserved.