To discuss the internal mass distribution in microbial fuel cell (MFC), a transient, two-dimensional model for single-chamber, air cathode MFC was developed in this work. This model was established by finite element method considering two kinds of microorganisms' growth, internal mass transfer and bio-electrochemical kinetics. The heterogeneous chemical components distribution in the anode chamber, the growth and spatial distribution of exoelectrogens and methanogens were discussed. The effect of biofilm porosity and external resistance on the electron transfer from redox mediator to the anode, microorganism growth and electricity generation performance in MFC was investigated. Simulation results revealed that the exoelectrogens and methanogens concentrations distributed heterogeneously with different biofilm porosities. Higher biofilm porosity was beneficial to final electron transfer step and had different impact on electricity generation at start-up and steady stage, respectively. Lower external resistances contributed to enhancing MFC performance. Our model should be helpful for the optimization of the design and operation conditions in MFCs.