A　new　flow-through　electrochemical　immunosensor　was　designed　for　sensitive　detection　of　alpha-fetoprotein　(AFP)　in　human　serum　by　using　nanogold-functionalized　magnetic　graphene　nanosheets　as　immunosensing　probes.　Initially,　amino　functionalized　magnetic　beads　were　covalently　immobilized　on　the　surface　of　graphene　oxide　nanosheets　(MGPs),　then　nanogold　particles　were　adsorbed　on　the　amino　groups　of　the　MGPs　to　construct　GoldMag　nanocomposites　functionalized　graphene　nanosheets　(GMGPs),　and　then　horseradish　peroxidase-anti-AFP　conjugates　(HRP-anti-AFP)　were　assembled　onto　the　surface　of　nanogold　particles　(bio-GMGP).　With　the　aid　of　an　external　magnet,　the　formed　bio-GMGPs　were　attached　onto　the　base　electrode　in　the　flow　system.　With　a　non-competitive　immunoassay　format,　the　injected　sample　containing　AFP　antigens　was　produced　transparent　immunoaffinity　reaction　with　the　immobilized　HRP-anti-AFP　on　the　bio-GMGPs.　The　formed　immunocomplex　inhibited　partly　the　active　center　of　HRP,　and　decreased　the　labeled　HRP　toward　the　reduction　of　H2O2.　The　performance　and　factors　influencing　the　performance　of　the　immunosensor　were　investigated　in　detail.　Under　optimal　conditions,　the　electrochemical　immunosensor　displayed　a　wide　working　range　of　0.01-200　ng　mL(-1)　with　a　low　detection　limit　CLOD)　of　1.0　pg　mL(-1)　AFP　(at　3s(B)).　Intra-　and　inter-assay　coefficients　of　variation　(CV)　were　below　10%.　In　addition,　the　methodology　was　validated　with　real　serum　samples,　receiving　a　good　correlation　with　the　results　obtained　from　commercially　available　electrochemiluminescence　automated　analyzer.　(C.)　2011　Elsevier　B.V.　All　rights　reserved.