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The exploration of low-cost, long-term stable, and highly electrochemically active cathode catalysts is important for the practical application of microbial fuel cell (MFC). In this work, a series of the 3D hierarchical porous Co–N–C (3DHP Co–N–C) materials are designed and synthesized by a metal-organic framework ZIF-67 as a precursor and SiO 2 sphere of different sizes as the hard template. The 3DHP Co–N–C-2 with 129 nm macropore exhibits excellent ORR performance in 0.1 M KOH solution with a half-wave potential of 0.80 V vs. RHE and superior durability than Pt/C (20%) due to the specific macropore-mesopore-micropore structure that exposes a large number of active sites and accelerates the electrolyte transport and oxygen diffusion. The MFC with 3DHP Co–N–C-2 as the cathode catalysts shows excellent performance with a maximum power density of 426.9 ± 7.87 mW m−2 and favorable durability after 50 d of operation. In addition, 16s rDNA results reveal the presence of different dominant electrogenic bacteria and different abundance of important non-electrogenic bacteria in the anode biofilm in MFCs using cathode catalysts with different ORR activity. And 3DHP Co–N–C-2 was found to be beneficial to the synergistic effect of electrogenic and non-electrogenic bacteria. This study explores electrocatalysts in terms of both electrocatalytic activity and anode microorganisms, providing new and comprehensive insights into the power generation of MFC. [Display omitted] • The 3DHP Co–N–C was synthesized by a hard template method. • The hierarchical porous structure of 3DHP Co–N–C facilitated electrocatalysis. • The microbial community in MFC with 3DHP Co–N–C favored electricity production. [ABSTRACT FROM AUTHOR]