The main reasons that make mankind look for new sources of energy are decreasing dependence on fossil fuels and reducing pollution. Wastewater treatment with the help of microbial fuel cells is an area where these two goals can be combined. Microbial fuel cells, in which microorganisms catalyze the oxidation of organic substances, represent a new and promising alternative for electricity generation. The creation of an efficient cathode in such systems is the important problem. It is evident that in these applications an oxygen (air) electrode is a promising cathode. In this study, the oxygen biocathode based on the laccase enzyme Pleurotus ostreatus HK-35 was developed and its electrochemical properties were studied depending on the immobilization method of the enzyme on the surface of a carbon-graphite electrode and the type of the electrolyte. It was experimentally established that laccase injection using a sol-gel matrix was the effective method for immobilizing laccase on the surface of a carbon-graphite electrode. It was shown that the more efficient operation of the laccase-based biocathode was observed in a phosphate-citrate (pH 4.0) buffer solution, i.e., in an acidic environment.