Methods　based　on　nanomaterial　labels　have　been　developed　for　electrochemical　immunosensors　and　immunoassays,　but　most　involved　low　sensitivity.　Herein　a　novel　class　of　molecular　tags,　nanogold-polyaniline-nanogold　microspheres　(GPGs),　was　first　synthesized　and　functionalized　with　horseradish　peroxidase-conjugated　thyroid-stimulating　hormone　antibody　(HRP-Ab　2)　for　sensitive　electrochemical　immunoassay　of　thyroid-stimulating　hormone　(TSH).　X-ray　diffraction,　confocal　Raman　spectroscopy,　scanning　electron　microscope　and　transmission　electron　microscope　were　employed　to　characterize　the　prepared　GPGs.　Based　on　a　sandwich-type　immunoassay　format,　the　assay　was　performed　in　pH　5.0　acetate　buffer　containing　6.0mmolL　-1　H　2O　2　by　using　GPG-labeled　HRP-Ab　2　as　molecular　tags.　Compared　with　pure　polyaniline　nanospheres　and　gold　nanoparticles　alone,　the　GPG　hybrid　nanostructures　increased　the　surface　area　of　the　nanomaterials,　and　enhanced　the　immobilized　amount　of　HRP-Ab　2.　Several　labeling　protocols　comprising　HRP-Ab　2,　nanogold　particle-labeled　HRP-Ab　2,　and　polyaniline　nanospheres-labeled　HRP-Ab　2,　were　also　investigated　for　determination　of　TSH　and　improved　analytical　features　were　obtained　by　using　the　GPG-labeled　HRP-Ab　2.　With　the　GPG　labeling　method,　the　effects　of　incubation　time　and　pH　of　acetate　buffer　on　the　current　responses　of　the　immunosensors　were　also　studied.　The　strong　attachment　of　HRP-Ab　2　to　the　GPGs　resulted　in　a　good　repeatability　and　intermediate　precision　down　to　7%.　The　dynamic　concentration　range　spanned　from　0.01　to　20μIUmL　-1　with　a　detection　limit　(LOD)　of　0.005μIUmL　-1　TSH　at　the　3s　B　criterion.　Significantly,　no　significant　differences　at　the　0.05　significance　level　were　encountered　in　the　analysis　of　15　spiking　serum　samples　between　the　developed　electrochemical　immunoassay　and　the　commercially　available　enzyme-linked　immunosorbent　assay　(ELISA)　method　for　determination　of　TSH.　©　2012　Elsevier　B.V..