OBJECTIVE To explore electricity generation and dynamic characteristics of microbial community of microbial fuel cells (MFC) started-up with mixed sludge of aerobic/anaerobic sludge. METHODS Single-chamber MFCs were constructed and characteristics of microbial community composition and structure were investigated by culture-independent microbial molecular techniques. RESULTS MFC started up successfully after three cycles' operation, and the maximum output voltage was up to 230 mV. The maximum power density reached 11.15 W/m3 at the outer resistance of 1656 Ω. The structure of microbial community on the anodic biofilm was different from that of seed sludge and microbial diversity reduced. The dominant microbial groups on anodic biofilm were Betaproteobacteria (24.90%), Bacteroidetes (21.30%), Firmicutes (9.70%), Gammaproteobacteria (8.50%), Deltaproteobacteria ( 7.90%), Chloroflexi (4.20%) and Alphaproteobacteria (3.60%). The biofilm-forming microbial genera Zoogloea and Acinetobacter accounted for 5.00% and 3.90% of total community. The abundance of electricity-producing bacteria Geobacter spp. increased from 0.60% in mixed sludge to 2.60% on the biofilm. CONCLUSION Dominant microbial populations in mixed sludge were selected by long-term acclimation and finally a beneficial microbial group was built on the anodic biofilm. The populations group was helpful to form a functional and active biofilm, which consequently benefited to produce electricity under anaerobic condition by fermenting organic matter.