An enzymatic biofuel cell cathode was elaborated by nano-structuration and functionalization of graphite using two consecutive plasma treatments before immobilization of laccase as a biocatalyst to perform oxygen reduction reaction (ORR) by direct electron transfer ((DET). Nano-structuration of graphite surfaces by synthesis of vertical graphen-like carbon nanowalls (CNWs) was performed by microwave excited plasma enhanced chemical vapor deposition (PECVD). Atmospheric pressure plasma jet (APPJ) system was then used to functionalize chemically the CNWs surface via a rapid and dry route. A partial fractional design evidenced two significant parameters of the APPJ treatment in terms of ORR current density: the treatment time and the inter-distance between the plasma jet and the surface of the carbon material. With the optimized APPJ treatment parameters, a four-fold enhancement of the ORR current density (-440 ± 70 µA/cm2) could be obtained on CNWs, compared to graphite electrodes. The best current density obtained was about −1 mA/cm2 with oxidized laccase covalently immobilized on the electrode without any agitation of the solution, confirming the main importance of enzyme orientation on the electrode surface for efficient DET transfer, the relevance of electrode nanostructuration and the advantage of covalent linking of the protein on the electrode surface.