The use of ligninolytic enzymes in biotechnological applications requires a highly effective production system, with sufficient amounts of the enzymes to be applied in experimental research and herein after at large-scale operations. To reach this final goal, we propose scale-up of ligninolytic production of one of the most well-known enzymes, Manganese Peroxidase (MnP), by Bjerkandera sp. BOS55. Taking into account previous results obtained in shaken flask cultures, MnP production was attempted in stirred fermenters of 2, 10 and 50 l, with levels of activity comparable to those obtained at a lower scale. Additionally, environmental factors as agitation rate, fungus immobilisation and use of buffer were evaluated to maximise MnP production. A fed-batch strategy was proved to reactivate MnP production and to maintain MnP activity for a longer period of time. Operational parameters, such as pH and Redox potential, monitored along the fermentation were found to be useful indicators of MnP production. These variables experimented drastic changes at the MnP peak production, signalling the right moment to collect the enzyme.