N, N-dimethylformamide (DMF) has been widely used as an organic solvent in chemical industries. In this study, the assessment of sustainability and carbon neutrality was investigated by a lab-scale up-flow anaerobic sludge blanket (UASB) reactor for the treatment of high-strength DMF-containing industrial wastewater. The UASB was operated at a limited organic loading rate (OLR) of 1.57–9.59 COD kg/m/d with the HRT shortened from 48 h to 8 h, and succeeded in an excellent removal efficiency of over 95%. The CO emission and bio-energy production for the treatment of DMF-containing wastewater were estimated and calculated using empirical equations. Since the conventional activated sludge (CAS) process is unable to recycle energy and reduce CO emission, the UASB has demonstrated remarkable superiorities for the treatment of synthetic DMF-containing wastewater by obtaining a positive net energy potential of 0.061 kWh/m. The UASB significantly reduced 6.18 kg/m of the CO emission in comparison with the CAS process. The UASB process also exhibited the remarkable economic potential by saving the electricity consumption of 12.20 kWh/m for treating the real wastewater. The application of the UASB in a wastewater treatment plant dealing with real DMF-containing wastewater would gain 4.74 million Chinese Yuan (CNY) which was supposed to pay for the electricity expense every year. The significant findings of this study are clear evidence of the promising potential and feasibility of the UASB process, also facilitate the popularization of the concept of carbon neutrality in wastewater treatment.