Cell-immobilization technology has long been considered as an important tool for microbial fuel cell (MFC) design. For the first time, a direct comparison between MFCs catalyzed by freely-suspended cells, surface-attached cells, and matrix-embedded cells for simultaneous electricity generation and distillery wastewater treatment in both batch and continuous operation modes was systematically performed. The results indicated that MFCs catalyzed by surface-attached cells and matrix-embedded cells had better chemical oxygen demand (COD) removal and power production efficiencies than those catalyzed by freely-suspended cells. The normalized energy recovery (NER), or power divided by the amount of COD consumed, was found to be decreasing with increasing COD concentration in all three different setups. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that the structure of microbial community was influenced by the mode of operation, COD concentration and cell immobilization methods of the MFC. Nevertheless, surface-attached cells and matrix-embedded cells had a higher similarity than freely-suspended cells regardless of the mode of operation. The information obtained here will be an important reference for future design and application of MFCs for simultaneous wastewater treatment and power production.