Understanding the role of extracellular polymeric substances (EPS) in the microbial reduction of uranium accelerated by mediating materials is crucial for enhancing the bioremediation of uranium-contaminated wastewater. In this study, biochar- and magnetite-loaded Desulfovibrio vulgaris UR1 exhibited significantly higher uranium reduction efficiency, with increases of 1.52 and 1.44 times respectively within one day. After loading with mediating materials, the charge transfer resistance of EPS was reduced, facilitating the extracellular electron transfer process. The increase of redox components, such as aromatic compounds and flavins, in EPS explained the enhanced extracellular electron transfer capacity. Moreover, the higher α-helix content in extracellular proteins could promote electron hopping. Proteomics analysis showed that extracellular proteins involved in iron-sulfur cluster binding, oxidoreductase activity, and electron transfer were significantly up-regulated, which facilitated the rapid microbial reduction of uranium. These findings provide valuable insights into the in-depth development of bioremediation technology for uranium-contaminated wastewater.