Dual chambered continuous up-flow microbial fuel cell (MFC) was used to check the effect of controlled temperature and addition of different hydrogen peroxide (H2O2) doses on bioelectricity production. MFC-1 and MFC-2 showed 77 and 89% of COD removal efficiency, respectively, while same amount 13.4% of coulombic efficiency under continuous operation mode were produced by both reactors. Oxygenation of cathode chambers of both MFC with 5 mL of H2O2 resulted in higher values of potential difference and current, 1100 mV and 0.6 mA in MFC-1 and 674 mV and 0.32 mA in MFC-2. Higher power density of 166 Pd·cm−2 was produced by reactor 1 than 75 Pd·cm−2 from reactor 2. Result showed that control temperature of 35°C had lowered down the bioelectricity production while increased the COD removal. The use of H2O2 for oxygenation was found to improve the voltage and current production and stability of MFC.