Enzymatic biofuel cells (EBCs) have been extensively studied over the last ten years in view of their application as implantable devices [1,2] as they have been shown indeed to reach the power required for small electronic devices. In EBCs, redox reactions occurring at the cathode and the anode are catalysed with the help of enzymes. Multicopper oxidases (MCO) such as laccases are well established biocatalysts for the oxygen reduction reaction (ORR) occurring at the cathode at low overpotentials, either by direct electron transfer (DET) [3] or by mediated electron transfer (MET). Unlike DET, MET exploits freely diffusing redox mediators in order to permit and speed-up the electron transfer between the enzyme active site and the electrode. In order to avoid the use of redox mediators, most of which are potentially toxic, numerous efforts were devoted to the study of EBCs working by DET. This latter was observed mainly with redox proteins whose redox active centers are close to the protein surface, which is the case of several MCOs. However, the optimization of the electronic communication between the electrode and the enzyme is a priority for the DET efficiency, which implies that the enzyme orientation and consequently the immobilization method (either electrostatic, based on hydrophobic interactions or covalent bonding) of this enzyme on the electrode surface are both crucial. Among electrode materials allowing the immobilisation of enzymes, numerous carbon materials (porous carbons, carbon nanotubes, carbon black) have been preferentially used because of their low cost, their good conductivity, and their large electroactivity window, even though none of them possesses the superficial amine or carboxylic acid groups that are necessary for covalent grafting of enzymes. As a consequence, different methods of functionalization were developed in order to enrich the surface chemistry of carbon materials, such as diazonium chemistry, amination, chemical oxidation, π-stacking or plasma treatment. In this contribution, Laccase type purified enzymes obtained from Trametes versicolor were covalently grafted for the first time on amorphous carbon nitride materials (a-CNx), used thus here as biocathode materials [4]. This was possible thanks to the natural presence of amines on its surface or to the electrochemical production of carboxylic acid type surface functional groups. The aim was here to control the orientation of the covalently immobilized redox enzymes so as to evaluate its impact on DET efficiency and therefore on ORR kinetics. For this purpose, the a-CNx materials were preliminarily deposited either on rough graphite-based electrodes in view of electrochemical (i.e. cyclic voltammetry or EIS) measurements related to oxygen reduction, or on an atomically smooth silicon wafer allowing AFM investigations. This latter type of substrate permitted indeed the use of amplitude modulation-atomic force microscopy (AM-AFM or tapping mode AFM) imaging and phase imaging of AFM (PI-AFM). These working modes of AFM were used, as well as Scanning Electron Microscopy (SEM-FEG), and Xray Photoelectron Spectroscopy (XPS) so as quantify the enzyme deposit and to characterize its morphology at the surface of the resulting biocathodes as a function of the enzyme immobilisation method. The ultimate goal was to correlate cathodic current density values resulting from ORR with parameters extracted from EIS measurements, in order to develop a better understanding of the influence of the covalent grafting method of Laccase on DET with the electrode, and consequently on the kinetics of ORR. References [1]A. de Poulpiquet et al., ChemElectroChem, 1 (2014) 1724-1750. [2]S. Cosnier et al., Electrochem. Commun., 38 (2014) 19-23. [3]S.M. Jones et al., Cell. Mol. Life Sci., 72 (2015) 869e883. [4] A. Blout et al., Electrochim. Acta, 277 (2018) 255-267.