In this study, a new process was developed for manganese peroxidase (MnP) production by Phanerochaete chrysosporium under an agitated and aerated cultivation condition. It was found that change of the inoculum from spore suspension to pellets resulted in enhanced MnP production of 200 U/L in rotated shake flasks. Several additives, including Pluronic F68, Tween 80, and PEG8000, significantly increased the enzyme production. With an optimal concentration in 125 mL flasks, Pluronic F68 increased MnP productivity by 180%. Moreover, successful enzyme production was achieved in a 5-L fermentor at an agitation speed of 300 rpm with the addition of 0.1% Pluronic F68.