A tubular bio-electro Fenton (BEF) system is designed in a field-scale groundwater application. An Fe-graphite felt (Fe@GF) cathode with optimal iron loading was used to prevent the generation of iron sludge. The Fe@GF cathode generates H2O2 through the 2-electron oxygen reduction reaction, facilitating the removal of sulfolane. For Fe@GF with an iron loading of 0.3 g/L, the reduction peak current reached −1.20 mA, which was 9.3 times that for GF. To optimize the performance of the BEF system, various external resistances were studied, and 10 Ω was found to yield the highest current density of 1034 mA/m2 and the highest H2O2 concentration of 8.9 mg/L. The presence of OH∙ was confirmed following the Fenton reaction, with 100 % removal of 50 mg/L of sulfolane. Microbial community analysis at an external resistance of 10 Ω revealed that electrochemically active bacteria (Clostridiaceae and Dysgonomonadaceae) were the dominant species. Low external resistance was found to favor in activation of the BEF process.