Fe₃O₄ nanoparticles (FNP) were implemented in two-stage anaerobic digestion to enhance the biogas production and waste sludge reduction in this study. The degradation and transformation of extracellular polymeric substances (EPS) and dissolved organic matters (DOM) under optimal FNP concentration during two-stage anaerobic process with heat pretreated waste sludge was investigated by analyzing the changes of soluble chemical oxygen demand (SCOD), carbohydrate and protein in EPS and DOM. FNP addition significantly enhanced volatile fatty acids (VFAs) production. The optimal dosage of FNP was 100 mg/L yielded 11.9 ml H₂/g VS hydrogen and 109.8 ml CH₄/g VS methane, which increased by 15.1% of hydrogen yield and 58.7% of methane yield compared with that of control. The maximum volatile suspended solids (VS) reduction was 46.9% with the addition of 100 mg/L FNP compared with that of control (34.3%). NH₄⁺-N production was inhibited with 20, 50, and 100 mg/L FNP addition comparing with control.