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　6.0　mmol　L-1　H2O2　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　20　mu　IU　mL(-1)　with　a　detection　limit　(LOD)　of　0.005　mu　IU　mL(-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.　(C)　2012　Elsevier　B.V.　All　rights　reserved.