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.