A　simple　and　sensitive　electrochemical　immunoassay　method　was　developed　for　the　detection　of　alpha-fetoprotein　(AFP)　in　human　serum.　The　immunoassay　is　based　on　the　use　of　an　AFP-functionalized　thionine/nanogold　multilayer　film　({Thi/AuNP}n)　on　a　single-walled　carbon　nanotubes　(CNT)-coated　gold　electrode　and　double-codified　gold　nanoparticles　with　horseradish　peroxidase-labelled　anti-AFP　antibodies　(HRP-anti-AFP-AuNPs).　With　a　competitive　immunoassay　format,　the　assay　was　performed　using　HRP-anti-AFP-AuNP　as　trace　and　H2O2　as　enzyme　substrate.　The　electrochemical　behavior　of　the　multilayer　{Thi/AuNP}n　films　was　studied.　The　maximal　signal　was　obtained　at　n　=　3　(i.e.　{Thi/AuNP}3).　Under　optimal　conditions,　the　obtained　electrochemical　responses　were　proportional　to　the　AFP　levels　in　the　sample.　The　dynamic　range　for　AFP　quantification　was　0.25-45　ng　mL　-1.　Both　the　intra-　and　inter-assay　coefficients　of　variation　were　less　than　9.5%.　The　proposed　assay　had　a　detection　limit　of　0.05　ng　mL　-1,　which　was　20-fold　vs.　10-fold　lower　than　that　obtained　using　monolayer　Thi/AuNP　film　vs.　conventional　HRP-labelled　anti-AFP　antibodies.　Importantly,　no　significant　differences　at　the　0.05　confidence　level　were　encountered　in　the　analysis　of　clinical　serum　samples　between　the　proposed　immunoassay　and　the　commercially　available　Roche　2010　Electrochemiluminescent　(ECL)　Automatic　Analyzer　for　AFP　determination.　©　2010　The　Royal　Society　of　Chemistry.