Abstract: Against the backdrop of synergistic advancement between the dual carbon strategy and circular economy, biogas projects serve as the core vehicle for resource utilisation of organic waste and renewable energy supply. Enhancing the efficiency of anaerobic digestion—the core process—is therefore paramount. Anaerobic digestion relies on the synergistic metabolism of microbial communities, including acidogenic and methanogenic bacteria. However, the impact and underlying mechanisms of oxygen—inevitably introduced during the open-feed process of large-scale biogas projects—on methanogenic efficiency remain poorly understood. This study addressed the dynamic characteristics of oxygen exposure during actual feedstock introduction. Using fermented food waste liquor as inoculum (pH 6.8, total solids 17.8 g/kg, total volatile solids 8.32 g/kg, VS/TS 46.74%), with glucose as the carbon source and an organic loading rate of 2 g VS/L. Five oxygen concentration gradients were established: 0% (control group), 0.4%, 2%, 4% and 8%. Batch anaerobic digestion experiments were conducted in 100-millilitre glass pressure vessels (effective volume 50 millilitres), with three replicates per group. Using a multi-dimensional approach involving methane yield monitoring, volatile fatty acid (VFA) content analysis, dissolved oxygen and redox potential monitoring, microbial community analysis and metagenomic sequencing, this study systematically investigated the patterns of oxygen’s influence on anaerobic methanogenesis and its underlying mechanisms, and proposed targeted regulatory strategies. The results indicate that increased oxygen concentration significantly inhibits methane production efficiency, with the degree of inhibition increasing as oxygen concentration rises. The cumulative methane yield in the control group was 169.31 ml/gVS, whilst that in the 2% oxygen group was 161.43 ml/gVS, representing a 4.23% decrease compared to the control group. The methane yield in the 4% oxygen group was 149.76 ml/gVS, representing an inhibition rate of 11.97%. The 8% oxygen group produced only 94.68 ml/gVS, with an inhibition rate of 41.67%. The 0.4% oxygen group produced 173.06 ml/gVS, demonstrating a slight promotion of methane production. Elevated oxygen concentrations directly lead to the accumulation of dissolved oxygen and increased redox potential within anaerobic digestion systems. Both parameters demonstrate significant dependence on oxygen concentration gradients, constituting key environmental factors for oxygen stress-induced suppression of methane production. Conversely, low oxygen concentrations are rapidly consumed by system microorganisms, maintaining dissolved oxygen and redox potential at optimal levels.VFA metabolic analysis revealed differential effects of oxygen stress on fatty acid degradation: no significant impact on propionate degradation, but mild inhibition of acetate degradation, with acetate accumulation in the 8% O₂ group increasing by 115.80% compared to the control; while significantly inhibiting butyrate degradation, with butyrate accumulation in the 2% and 8% O₂ groups exceeding the control group by 343.94%. This indicates that oxygen stress indirectly inhibits methane production by impeding the degradation of key intermediate metabolites. Microbial community structure analysis revealed distinct stress response characteristics: At the bacterial level, the genus Defluviitoga emerged as the dominant phylum (abundance 17.53%–17.84%), with its abundance slightly increasing with rising oxygen concentration. Conversely, the abundance of the genus Acetomicrobium, known for its efficient glucose degradation and acid production, exhibited a decreasing trend (control group 6.68% > 2% O₂ group > 8% O₂ group 3.61%); In the 8% O₂ group, the facultative anaerobe genus Thiopseudomonas became highly enriched. This strain possesses a comprehensive defence system comprising superoxide dismutase (SOD) and catalase (CAT), enabling it to withstand oxygen toxicity and form an oxidative protective barrier within the anaerobic system. Within the archaeal community, the dominant methanogen Methanoculleus maintained stable abundance (71.61%–73.71%). This stability correlates with Thiopseudomonas' protective function and mutualistic metabolism with Defluviitoga and Acetomicrobium. Metagenomic functional analysis further elucidated the molecular mechanisms: under oxygen stress, key functional genes in the VFA-to-methane metabolic pathway (e.g., tpiA, fbaB, porB, ackA) exhibited significantly reduced abundance, directly impairing organic matter conversion efficiency. while antioxidant enzyme-encoding genes (SOD1, SOD2, katE) exhibited positive correlation with oxygen concentration, reflecting adaptive strategies within the microbial community. To mitigate oxygen suppression effects, experiments with exogenous antioxidant enzyme supplementation demonstrated optimal regulatory effects from combined SOD and CAT addition: under 2% O₂ conditions, methane yield increased by 6.69% compared to the control group; under 8% O₂ hyperoxic stress, methane yield increased by 14.03% compared to the 8% O₂ group. This study systematically elucidates the influence patterns of varying oxygen concentrations on anaerobic methane production under dynamic oxygen exposure. By clarifying the multi-level mechanisms involving microbial community restructuring, regulatory mechanisms of functional gene expression, and accumulation of intermediate metabolites. The proposed combined exogenous antioxidant enzyme supplementation technique fills a gap in research on methanogenesis mechanisms under dynamic oxygen exposure, providing crucial theoretical support and practical technical solutions for enhancing biogas plant operational stability and energy conversion efficiency.