Microbial electrosynthesis system (MES) has recently been shown to be a promising alternative way for realizing in-situ and energy-saving synthesis of hydrogen peroxide (H2O2). Though promising, the scaling-up feasibility of such a process is rarely reported. In this study, a 20 L up-scaled two-chamber MES reactor was developed and investigated for in-situ and efficient H2O2 electrosynthesis. The maximum H2O2 production rate of 10.82 mg L-1 h-1 and cumulative H2O2 concentration of 454.44 mg L-1 within 42 h were obtained with an input voltage of 0.6 V, cathodic aeration velocity of 0.045 mL min-1 mL-1, 50 mM Na2SO4, and initial pH of 3. The electrical energy consumption regarding direct input voltage was only 0.239 kWh kg-1 H2O2, which was further much lower compared with lab-scale systems. The obtained results suggested that the future industrialization of MES technology for in-situ synthesis of H2O2 and further application in environmental remediation have broad prospects.