N,N-dimethylformamide (DMF) is widely used as an organic solvent in the chemical industry. This study aimed to assess the treatment of high-concentration DMF-containing industrial wastewater using a lab-scale up-flow anaerobic sludge blanket (UASB) reactor. The UASB reactor operated within a limited range of organic loading rates (OLR) of 1.57~9.59 kg COD/(m3·d), with the hydraulic retention time (HRT) reduced from 48 hours to 8 hours. The reactor demonstrated excellent removal efficiency, exceeding 95%. CO2 emissions and bio-energy production during the treatment of DMF wastewater were estimated and evaluated using empirical equations. Compared to the conventional activated sludge (CAS) process, the UASB process exhibited significant advantages in terms of energy recovery and CO2 emission reduction. It achieved a positive net energy potential of 0.061 kW·h/m3 and substantially reduced CO2 emissions by 6.18 kg/m3. The results of this study provide evidence of the potential and feasibility of the UASB process for DMF waste treatment, while also contributing to the promotion of the carbon neutrality concept in wastewater treatment.