Direct electron transfer (DET) type biocatalysis was accomplished for Trametes hirsuta laccase (ThL) on a glassy carbon (GC) electrode by immobilizing laccase into a well-designed dual-layer architecture of poly(3,4-ethylenedioxythiophene) (PEDOT). PEDOT films were subsequently deposited on a GC electrode via electropolymerization, with NO3− as the counterion for the first accommodation layer and poly(styrene-sulfonate) anions (PSS−) for the second capping layer. The enzyme (ThL) was cast on top of the accommodation layer (PEDOT-NO3), and then the capping layer (PEDOT-PSS) was electrodeposited to entrap ThL between the layers. This enzyme electrode is reported to be able to promote DET between ThL and the GC electrode and catalyze the reduction of O2 into water. The influence of fabrication parameters on the enzyme electrode performance was investigated through chronoamperometric measurements. The investigated parameters included different combinations of PEDOT films, ThL loading, and the thicknesses of both PEDOT layers. As a representative, one optimized dual-layer-architecture enzyme electrode of PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) performed fairly good reproducibility and operational stability. Its pH profile exhibited a bell-shape with an optimal pH in the range of 3.0−3.5. The influences of ionic strength and addition of a nonionic surfactant into the buffer solution on the enzyme electrode performance were also studied to obtain information about the DET mechanism of ThL in the dual-layer architecture. On the basis of the information obtained from different characterizations, π−π interaction between the PSS− ions and the hydrophobic substrate-binding pocket in the vicinity of the T1 Cu site was proposed to result in a favorable location of the conducting polymer chain close to the T1 Cu site and thus facilitate DET of ThL within this particular architecture. The applicability of this approach to various electrode materials is also underlined, which makes it a favorable approach to construct an O2-consuming cathode for biofuel cells.