Iron-based oxide catalysts on biomass carbon fibers can jointly use their respective advantages of chemical composition and structure to develop catalysts for the oxygen reduction reaction (ORR). In this study, carbon-coated Fe3O4@cotton fiber biomass carbon (C@Fe3O4@CFBC) was prepared as an ORR catalyst through hydrothermal growth and calcination treatment for air cathode microbial fuel cells (MFCs). The carbon coating layer enhanced the O2 adsorption energy, decreased the overpotential, and promoted surface activation of the Fe3O4 catalyst. The carbon coating layer can modulate the surface electronic structure of the Fe3O4 catalyst with C–O–Fe bonding and improve the electrical conductivity to accumulate electrons on the Fe3O4 surface to enhance the ORR performance. The CFBC supported Fe3O4 nanoparticles to prevent severe aggregation and expose more effective surface-active sites. The C@Fe3O4@CFBC catalyst achieved a high positive half-wave potential (0.801 V) and low Tafel slope (73.7 mV·dec−1) compared with the Pt/C catalyst (0.877 V and 94.3 mV·dec−1). The assembled air cathode MFCs achieved a desirable maximum power density (1059 ± 16 mW·m−2) based on 1266 ± 94 mW·m−2 of Pt/C MFC devices. This work provides a strategy for designing highly active non-precious metal catalysts for air cathode MFCs.