Microbial Fuel Cells (MFCs) represent an environmentally-friendly approach to generating electricity, but the need to study variation parameters to find improvement conditions has been an important challenge for decades. In this study, a single-chamber MFC was designed to investigate the key parameters such as the concentration and type of bacteria, chamber temperature, electrode spacing, and substrate rotation speed that affected the performance of MFCs. Therefore, two types of bacteria, Shewanella oneidensis (S.one) and Escherichia coli (E. coli), were compared as microorganisms. Then, the function of MFC was investigated under the following condition: three temperatures (30 ℃, 45℃, and 60℃), three bacterial concentrations (0.5% (v/v) (4.5 mg/ml), 1% (v/v) (9mg/ml), and 1.5% (v/v) (13.5mg/ml)), electrode distances (2 cm, 3 cm, 4cm), and substrate speeds (100 rpm, 150 rpm, 200 rpm). Ultimately, (S.one) bacteria, a chamber temperature of 45 ℃, a bacterial concentration of 1% (v/v) (9mg/ml), a cathode-anode spacing of 3 cm, and a rotation speed of 150 rpm proved to be the most efficient parameter settings for the constructed microbial fuel cell. The maximum voltage and highest power density were 486.9 mV and 9.73 mW/ , respectively, with a resistance of 7500 ohms. These results are meaningful for determining and improving important parameters in an MFC device.