The bio-natural gas industry is currently experiencing rapid development in China. Bio-natural gas is purified from biogas, which is produced by anaerobic fermentation of biomass. China′s coastal regions possess abundant seaweed resources, a type of biomass with great potential for biogas production. Therefore, this research investigated biogas production from seaweed through anaerobic fermentation, with the aim of effectively utilizing seaweed and recycling biogas as a renewable energy source. Nano-magnetite was added to the anaerobic reactors to explore the potential to enhance biogas production. The results showed that nano-magnetite could accelerate the degradation of biomass and the production of methane. The removal efficiencies of total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) in nano-magnetite reactors increased by about 20% and 12%, respectively, compared with the control reactor, which showed removal efficiencies of 51.4% and 42.5%, respectively. Methane production in nano-magnetite reactors more than doubled, with the highest methane production of 1 320 mL at a nano-magnetite concentration of 2.0 g/L. Nano-magnetite enhanced the stability of the anaerobic system and promoted a quicker recovery to neutral pH following acidogenic fermentation of kelp. The pH level in nano-magnetite reactors was always higher than 6.2 and increased gradually to 7.5, while that in the control reactor decreased below 6.0 and then recovered slowly. Analysis of the microbial community structure indicated that nano-magnetite significantly accelerated the enrichment of electroactive microorganisms and facilitated direct interspecies electron transfer (DIET) in methane production. Scanning electron microscopy (SEM) showed that microorganisms and nano-magnetite adhered to each other in nano-magnetite reactors, which was beneficial for DIET to occur among electroactive microorganisms and between electroactive microorganisms and nano-magnetite during the anaerobic methane production process. High-throughput sequencing analysis showed that the electroactive microorganisms were distinctly enriched in nano-magnetite reactors. The abundance of norank_Anaerolineaceae (31.3%, 43.4%, 47.0%, and 42.1% for 0.2 − 5.0 g/L nano-magnetite); norank_Bacteroidetes_vadinHA17 (2.0%, 9.3%, 7.6%, and 7.8%); Leptolinea (2.6%, 7.3%, 6.7%, and 9.1%); Longilinea (2.9%, 4.6%, 4.8%, and 4.7%); Dechloromonas (2.3%, 2.0%, 1.2%, and 0.9%); and unclassified_Anaerolineaceae (9.2%, 8.9%, 8.5%, and 7.1%) all increased compared with the control reactor (17.3%, 1.4%, 1.6%, 3.2%, 0.6%, and 2.0%). Additionally, nano-magnetite increased the abundance of the Methylene-H4MPT reductase regulator gene (mer) and the heterodisulfide reductase regulator gene (hdrABC) in methanogens, enhancing the metabolic pathway of DIET methanogenesis. The abundance of mer was 4.57E−04, 5.45E−04, 5.75E−04, and 5.50E−04, and hdrABC was 3.69E−05, 1.06E−04, 1.04E−04, and 1.42E−04, while these values in the control reactor were 2.22E−04 and 6.48E−05, respectively. These findings suggest that nano-magnetite significantly enhances biogas production from seaweed biomass. This technology holds promise for further development to support the advancement of the biogas industry and contribute to achieving the "dual carbon" goal in China.