It is crucial to study the bacterial attaching behavior for revealing information of electrochemically active biofilm on the interface of liquid/electrode in microbial fuel cells (MFCs). In the current study, mixed-culture from waste water as model bacteria was investigated for gaining more sight onto relationship between current and biomass or onto bioadhesion mechanism at the molecular level using Electrochemical Quartz Crystal Microbalance (EQCM) and Attenuated Total Reflection-SEIRAS (ATR-SEIRAS) combined with electrochemistry. From the frequency shift using Sauerbrey equation via EQCM the maximum cells mass of 11.5 μg/cm 2 was estimated for the mature biofilm. Notably, the highest current density of 110 μA/μg·cm 2 occurred before maximum biomass coming, which indicated that mature biofilm may not be an optimal state for enhancing power output of MFCs. Furthermore, the sensed biomass attached on electrode for mature biofilm will be constant for more than 40 h * Corresponding author, On the other hand, using ATR-SEIRAS techniques the obvious adsorbed water structure change during biofilm formation on electrode surface was observed and showed that the absorption bands linked to bacterial adsorption increased. It can be concluded that water adsorption accompanies the adsorbed bacteria and the cells number attaching on the electrode increased with time. Especially, the direct contact of bacteria and electrode via outer-membrane protein can be confirmed via series spectra at amideⅠand amideⅡ modes and water movement from negative bands displacing by adsorbed bacteria. Our study provided supplementary information about the interaction between the microbes and solid electron acceptors beyond traditional electrochemistry. It is expected to explore more information on electrochemically active microbial kinetics for improvement of their competence and understanding of attaching mechanisms in nature using our approach.