Understanding how environmental factors affect the bioelectrode efficiency and stability is of uttermost importance to develop high-performance bioelectrochemical devices. By coupling fluorescence confocal microscopy in situ to electrochemistry, this work focuses on the influence of the ionic strength on electro-enzymatic catalysis. In this context, the 4 e-/4 H+ reduction of O2 into water by the bilirubin oxidase from Myrothecium verrucaria (MvBOD) is considered as a model. The effects of salt concentration on the enzyme activity and stability were probed by enzymatic assays performed in homogeneous catalysis conditions and monitored by UV-vis absorption spectroscopy. They were also investigated in heterogeneous catalysis conditions by electrochemical measurements with MvBOD immobilized at a graphite microelectrode. We demonstrate that the catalytic activity and stability of the enzyme both in solution and in the immobilized state at the bioelectrode were conserved with an electrolyte concentration of up to 0.5 M, both in a buffered and a non-buffered electrolyte. Relying on this, we used fluorescence confocal laser scanning microscopy coupled in situ to electrochemistry to explore the local pH of the electrolyte at the vicinity of the electrode surface at various ionic strengths and for several overpotentials. 3D proton depletion profiles generated by the interfacial electro-enzymatic reaction were recorded in the presence of a pH-sensitive fluorophore. These concentration profiles were shown to contract with increasing ionic strength, thus highlighting the need for a minimal electrolyte concentration to ensure availability of charged substrates at the electrode surface during electro-enzymatic experiments.