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). Although 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. 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 3. The electrical energy consumption regarding direct input voltage was only 0.239 kWh kg−1 H2O2, which was further much lower compared with laboratory-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. © 2021 The Author(s)