This paper describes the development of an amperometric biosensor based on galactose oxidase (GAOx) immobilization within a laponite clay film deposited on Carbon Screen-Printed Electrodes modified by electrodeposited Prussian Blue and coated with poly-(O-phenylenediamine) (PPD/PB/CSPEs). Amperometric performances of GAOx@laponite/PPD/PB/CSPEs bioelectrodes were determined using several GAOx substrates. Using these modified electrodes the reduction of enzymatically generated hydrogen peroxide was performed at −0.2 V vs. Ag-AgCl. In an initial attempt, E.Coli transketolase activity on its immobilized form was followed using a bienzymatic GAOx-TK biosensor.