An electrochemical biointerface of 3D graphene aerogel (3DGA) decorated with Au nanoparticles (AuNPs) was fabricated to immobilize Cytochrome c (Cyt c) for H2O2 detection. Morphology and surface characterization of 3DGA-AuNPs reveal a successful formation of 3D-networked structure that can enhance conductivity and effectively immobilize enzyme. The immobilized Cyt c retained its bioactivity and realized the direct electron transfer on electrode owing to the biocompatibility of AuNPs and large surface area of 3DGA. The as-prepared 3DGA-AuNPs/Cyt c/GCE exhibited a pair of well-defined redox peaks of Fe-III/II redox couple of Cyt c and revealed excellent catalytic activity toward H2O2 with high sensitivity of 351.57 mu A.mM(-1).cm(-2). This biosensor was further successfully used to real-time detection of trace amount of H2O2 released from living cells, indicating the promise for practical application.