Abstract: The application of livestock and poultry manure in microbial fuel cell (MFC) technology can not only treat the manure but also generate bioelectricity. However, the presence of refractory organic matters significantly limit the electricity generation and degradation efficiency of MFC. A substrate-enhanced MFC system was constructed, and sophorolipid, lysozyme, and 2-bromoethanesulfonate were used as substrate additives, respectively. Voltage collection, organic matter concentration test, and 16S rRNA were comprehensively adopted to systematically compare and explore the key parameters, such as the electricity generation efficiency of MFC, the degradation of organic matter in manure, and the changes in microbial community structure. The results showed that compared with the blank control group, 2-bromoethanesulfonate could enhance the electricity generation performance of the MFC system, shorten the system start-up time by 42.3%, and increase the system voltage by 23.1%. Lysozyme could significantly improve the degradation of livestock and poultry manure by enhancing the hydrolysis performance of manure. When the addition amount was 0.1 g/g (calculated by total solids), the removal rate of total chemical oxygen demand of the system reached 78.6% in about 20 days. The addition of 2-bromoethanesulfonic acid and lysozyme markedly increased the abundance of Proteobacteria and Actinobacteria in the system, significantly promoting the electron transfer at the anode and the degradation of livestock and poultry manure. In conclusion, 2-bromoethanesulfonic acid can be used as an optimal additive to comprehensively improve the electricity generation efficiency, microbial enrichment effect, and degradation effect of livestock and poultry manure in the system, providing a technical reference for the subsequent degradation of livestock and poultry manure by MFC.