A　new　electrochemical　immunosensing　protocol　for　sensitive　detection　of　alpha-fetoprotein　(AFP,　as　a　model)　in　human　serum　was　developed　by　means　of　immobilization　of　horseradish　peroxidase-anti-AFP　conjugates　(HRP-anti-AFP)　onto　graphene　and　nanogold-functionalized　biomimetic　interfaces.　The　low-toxic　and　high-conductive　graphene　complex　provided　a　large　capacity　for　nanoparticulate　immobilization　and　a　facile　pathway　for　electron　transfer.　With　a　one-step　immunoassay　format,　the　antigen-antibody　complex　was　formed　between　the　immobilized　HRP-anti-AFP　on　the　electrode　and　AFP　in　the　sample.　The　formed　immunocomplex　was　coated　on　the　electrode　surface,　inhibited　partly　the　active　center　of　HRP,　and　decreased　the　catalytic　reduction　of　HRP　toward　the　enzyme　substrate　of　H2O2.　Under　optimal　conditions,　the　decrease　of　reduction　currents　was　proportional　to　AFP　concentration,　and　the　dynamic　range　was　1.0-10　ng/mL　with　a　relative-low　detection　limit　(LOD)　of　0.7　ng/mL　AFP.　Intra-　and　inter-assay　coefficients　of　variation　(CVs)　were　less　than　10　%.　The　assay　was　evaluated　for　clinical　human　serum　samples,　including　8　(possible)　patients　with　hepatocarcinoma　and　3　normal　human　sera.　Correct　identification　of　negative/positive　samples　and　perfect　accordance　with　results　from　Elecsys　2010　Electrochemiluminescent　Automatic　Analyzer　as　a　reference　was　obtained.　Importantly,　the　graphene　and　nanogold-based　sensor　provided　a　promising　platform　for　the　detection　of　other　biocompounds,　and　could　be　further　applied　for　development　of　other　potential　electrochemical　bio/chemosensors.　©　2010　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim.