As the foundation stone of bioelectrochemical systems (BESs), the maintenance of electroactive biofilm with good metabolic activity is essential to achieve high and stable performance. Since proton transport has significant impacts on the biofilm metabolic activity through the microenvironment pH, the investigation of proton transfer properties interior the biofilms is crucial for enhancing BES performance. Herein, the proton transport in electroactive biofilms is explored by in situ microelectrode measurements. It achieves a methodological innovation for measuring the diffusion coefficient in live biofilm under non-destructive conditions. The non-destructive measurement of diffusion coefficients in situ not only bring the results more closely to reality, but also enriches the knowledge of biochemical processes occurring and physicochemical information. Combined with the mass transport and electricity production mathematical model, it is demonstrated that the accumulation of protons in biofilm is a key factor in inhibiting the electricity generation. The efficient characterization of proton transfer interior the biofilm provides a realistic guidance for the construction high performance BES.