To achieve synchronous regulation of growth and lignocellulose degradation in Pleurotus ostreatus (PO-01) during fungal residue biorefining, we systematically evaluated O₂ gradients (5%, 20%, 40%) and N₂/CO₂ regarding mycelial development, lignocellulose degradation, and bioethanol potential. A total of 20% O₂ emerged as the critical threshold, balancing mycelial growth (which was faster than that under 5% O₂) and lignocellulose degradation (with lignin degradation rate reaching 15.29%). Metabolomics identified 53 aromatic derivatives related to lignin degradation, with their abundance correlating with actual lignin degradation rates. Meanwhile, it clarified the synergistic degradation mechanism and bioinformatics characteristics of key lignin-degrading enzymes and confirmed the AA9 gene associated with cellulose degradation at the molecular level. Measurements of polysaccharide content and ethanol yield revealed that the 20% O₂ environment led to a remarkably high ethanol yield of 101.90 L·ha⁻¹. In contrast, 5% and 40% O₂ concentrations not only reduced the polysaccharide content but also inhibited bioethanol production, highlighting O₂ as a crucial factor in regulating the synergy between growth and degradation. After comprehensive analysis, this study designated 20% O₂ as the optimal parameter for the integrated biorefining of fungal residues, offering a gas-phase solution to overcome industrial bottlenecks in biofuel production.