An O2-consuming cathode for biofuel cell applications consisting of Trametes hirsuta laccase (ThL) immobilized in an electropolymd. fine-tuned poly(3,4-ethylenedioxythiophene) (PEDOT) bilayer structure as the catalyst was studied. A NO3 --doped PEDOT layer with a relatively porous structure was used as the immobilization matrix on which the enzyme was added by soln. casting. A capping layer of polystyrene sulfonate, PSS--doped PEDOT was then electrodeposited on top of the 1st PEDOT layer to entrap the ThL between the layers. The PEDOT-NO3 -/ThL/PEDOT-PSS-enzyme electrode is reported to be able to promote direct electron transfer (DET) between ThL and the current collector and it catalyzes the redn. of O2 into H2O. The applicability of the PEDOT-NO3 -/ThL/PEDOT-PSS- enzyme electrode structure on ITO glass with a geometrical surface area of 1 cm2 as the electrode material was studied. The influence of different enzyme electrode fabrication parameters, such as the dopant ion used during electropolymn., different combinations of PEDOT films, the thickness of both PEDOT layers and ThL loading on the enzyme electrode performance were studied by chronoamperometric and cyclic voltammetric measurements. The optimum working pH for the enzyme electrode is in the pH range 3.0-3.5. No enhanced cell performance was recorded when 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was used as mediator compared to DET in the PEDOT bilayer structure.