A three chamber microbial fuel cells(MFC),employing escherichia coli (E. coil) as anode catalyst, was designed. Its electricity generation and ammonia nitrogen removal were investigated in mediator-less, new methylene blue (NMB)as mediator and neutral red (NR)as mediator conditions. The results show that mediators improved open circuit voltage(VOC)of MFC and reduced the time of reaching steady-state VOC. With NMB mediator, VOC of MFC achieved a stable value more easily. VOC of MFC with NR mediator was higher than that of MFC with NMB mediator. Meditators reduced denitrification time of MFC(ammonia nitrogen removal efficiency of 90%),besides, denitrification time of MFC with NMB was shorter. VOC was higher with NR as mediator, but VOC had slower attenuation and better stability with NMB as mediater.