Lignocellulosic biomass remains underutilized in anaerobic digestion (AD) due to its recalcitrant structure and limited microbial accessibility. This study investigated the combined effect of dilute-acid pretreatment and Fe3O4 nanoparticle supplementation on biogas production from wheat straw co-digested with cow dung. Wheat straw was pretreated with 1%–4% H2SO4, resulting in notable compositional alterations. Among the pretreatments, 2% H2SO4 increased cellulose content from 38.99% in the untreated straw to 43.63% (approximately 12% relative enrichment) while reducing lignin to 9.84%, indicating improved substrate accessibility. The optimized pretreated wheat straw was subsequently subjected to AD with Fe3O4 nanoparticle supplementation at concentrations of 0–50 mg/L. Characterization of Fe3O4 nanoparticles using XRD, FTIR, SEM, and TEM confirmed their crystalline nanoscale magnetite structure with favourable physicochemical properties for enhanced microbial electron transfer. Biogas production increased with nanoparticle dosage up to 30 mg/L, which produced the highest cumulative biogas yield (273.18 mL g-1 VS), representing an enhancement of approximately 37% over the control. Methane concentration also improved, reaching nearly 66% at this dosage. Kinetic analysis revealed that the modified Gompertz model best described the digestion process (R2 > 0.97), with the highest biogas potential (278.2 mL g-1 VS) and maximum production rate (19.2 mL g-1 VS d-1) observed at 30 mg/L Fe3O4. Overall, the integrated acid pretreatment and nanoparticle supplementation strategy significantly enhanced biogas production, demonstrating a practical and effective strategy for efficient lignocellulosic biomass valorisation through improved biomethane production.