•First scaled-up bio-electro-Fenton process for textile wastewater treatment.•High removal efficiency with low electric energy consumption in 20 L reactor.•Identified operating factors that can affect treatment performance at pilot-scale.•Successfully validated with six different dyes wastewaters in continuous mode.•Offers insight into development of bio-electro-Fenton process at industrial scale. Textile wastewater entering natural water bodies could cause serious environment and health issues. Bio-electro-Fenton (BEF) as an efficient and energy saving wastewater treatment technology has recently attracted widespread attention. So far, there is no research available on the scaling-up of BEF process. In this work, an innovative 20 L up-scaled BEF system was constructed for the treatment of methylene blue (MB) containing wastewater. The system was first tested in batch mode. The results showed that the system performance was majorly related to the operating parameters including initial MB concentration, catholyte pH and concentration, cathodic aeration rate, Fe2+ dosage, and applied voltage. At the optimal condition, 20 mg L−1 of MB was efficiently removed following the apparent first order kinetics. The corresponding rate constants for the decolorization and mineralization were 0.68 and 0.20 h−1, respectively. Furthermore, MB decolorization efficiency of 99% and mineralization efficiency of 74% were observed when the hydraulic retention time was 28 h in continuous mode. This work demonstrates the scaling-up potential of BEF for recalcitrant wastewater treatment.