The influence of a Reactive Brilliant Red X-3B (RBR X-3B) dye on the peroxidase isoenzyme of Phanerochaete chrysosporium was determined, and the biofilm structure in a white rot fungal continuous membrane bioreactor (MBR) was also investigated by scanning electron microscope (SEM). The variation of peroxidase isoenzyme and the decolorization rate in the continuous MBR were evaluated. The results showed that the 100 mg/L RBR X-3B could stimulate the production of the peroxidase isoenzyme in the shaking-flask culture. In addition, two new peroxidase isoenzyme bands with relative mobility (Rf) value of 0.27 and 0.28 appeared, but the activity was lower than the blank control of 11 d. In the continuous MBR, the system worked stably during the first 60 d, the main peroxidase isoenzyme bands existed and three new bands with Rf value of 0.10, 0.27, and 0.28 appeared. Meanwhile, the biofilm grew well and the average decolorization rate could reach 90.6%. But the bands of peroxidase isoenzyme decreased rapidly at day 65, only two bands with Rf value 0.24 and 0.26 existed, and the decolorization rate decreased to 78.3%. Therefore, 5 bottles of P. chrysosporium mycelial pellet were added into the MBR, and then the activity of the peroxidase isoenzyme and the decolorization rate had a slight recovery. Finally, the decolorization rate finally decreased to 75.2%. These results contribute to a comprehensive understanding of the variation of peroxidase isoenzyme and biofilm in continuous MBR by white rot fungi.