Dark fermentation of food waste for biohydrogen production can simultaneously achieve waste resource utilization and clean energy production. However, the widespread application of this technology remains constrained by challenges such as low substrate hydrolysis efficiency and suboptimal metabolic performance of functional microorganisms. This study evaluated the synergistic enhancement of biohydrogen production from food waste through dark fermentation by integrating thermal–alkaline (TA) pretreatment with varying concentrations (50, 100, 150, and 200 mg/L) of nickel–cobalt oxide nanoparticles (NiCo₂O₄ NPs), and the underlying mechanisms involved were systematically elucidated. The results demonstrated that individual TA pretreatment (pH 11, 70 °C, 1 h) and TA coupled with NiCo₂O₄ NPs (100 mg/L) significantly (p < 0.01) enhanced the cumulative biohydrogen yields of the food waste dark fermentation by 20.89% and 35.76%, respectively. Mechanism research revealed that TA pretreatment effectively facilitated the dissolution and hydrolysis of macro-molecular organics such as polysaccharides and proteins, thereby enhancing the bio-accessibility of fermentation substrates. The introduction of NiCo₂O₄ NPs further intensified the microbial biohydrogen-producing metabolism by augmenting enzymatic activity and enriching functional bacteria. NiCo₂O₄ NPs significantly (p < 0.001) enhanced the overall activity of hydrogenase by 95.10% compared to the control group (CG) by providing the cofactor of hydrogenase and accelerating electron transfer. Additionally, this synergistic strategy significantly (p < 0.01) increased the activities of hydrolases (e.g., protease and α-glucosidase), as well as key enzymes in acetate-type and butyrate-type fermentation pathways (e.g., acetate kinase and butyrate kinase), and enriched the biohydrogen-producing microbial community centered on Clostridium_sensu_stricto_1. This study systematically elucidated the synergistic strategy of TA pretreatment and NiCo₂O₄ NPs, which achieved dual-pathway reinforcement from substrate degradability to microbial metabolic activity. The findings are expected to provide theoretical support for developing efficient biohydrogen production technology from perishable organic solid waste.

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