Enzymatic biofuel cells are regarded as a promising method to meet the increasing demand for the power source. However, low output power is still a problem to be overcome in this field. Here, we constructed a glucose oxidase/horseradish peroxidase biofuel cell and for the first time systematically explored the role of oxygen solubility and mass transport in enhancing the power output of a biofuel cell combined with finite element analysis. It was confirmed that the increase of oxygen solubility led to higher electron transfer efficiency, which increased the maximum power density from 19.40 to 24.97 μW cm−2. Additionally, the main results show that the relative distance and position of the electrodes affect the mass transport between the cathode and anode, which in turn affects the performance of the fuel cell. Under the computed oxygen solubility of 1.919 ± 0.20 mM and the optimal structure, the biofuel cell fabricated on the carbon paper exhibited an open circuit potential of 0.41 V and maximum power density of 27.41 μW cm−2 and remained 82.4% of its original maximum power density after 20 days. This paper provides a facile and effective route for developing the output of enzymatic biofuel cells by enhancing dissolved oxygen concentration and mass transfer that would be of great significance to the structural design of enzymatic biofuel cell devices.