Based on self-assembled combination of graphene with Au nanoparticle-doped copper hexacyanoferrate (CuHCF) multilayers, a electrochemical biosensor for sensitive detection of hydrazine has been reported. Graphene was functionalized by wrapping with poly(diallyldimethylammonium chloride) (PDDA). Such polyelectrolyte modified graphene (PDDA-G) was water-soluble and possessed net positive charge. Based on electrostatic self-assembly, graphene multilayers modified electrode (ITO/G-M) was fabricated. After binding with mercaptopropionic acid stabled Au nanoparticles (MPA-AuNPs), three dimensional graphene-AuNPs electrode was obtained. CuHCF multilayers were then formed on AuNPs center by successive self-assembly and solution epitaxy. Due to the synergistic effect of graphene and AuNPs, the developed biosensor (ITO/G-M/CuHCF-M) exhibited fast and sensitive amperometric response for the determination of hydrazine in near physiological pH. The linear response for the determination of hydrazine ranged from 4.0 × 10-7 to 1.3 × 10-4 M with a detection limit of 7.2 × 10-8 M. The biosensor exhibited high reproducibility and stability resulted from simple and reproducible self-assembly methodology.