A　novel　class　of　redox-active　molecular　tags,　poly(o-phenylenediamine)-carried　nanogold　particles　(GPPDs),　was　first　synthesized　and　functionalized　with　horseradish　peroxidase-anti-prolactin　conjugates　(HRP-anti-PRL).　Thereafter,　a　specific　sandwich-type　electrochemical　immunoassay　was　designed　for　determination　of　prolactin　(PRL)　by　using　GPPD-labeled　HRP-anti-PRL　conjugates　as　molecular　tags　on　anti-PRL　antibody-modified　glassy　carbon　electrode.　Compared　with　pure　gold　nanoparticles　and　poly(o-phenylenediamine)　microspheres,　the　as-prepared　GPPDs　increased　the　surface　coverage　of　the　nanostructures,　and　enhanced　the　immobilization　amount　of　biomolecules.　Several　labeling　protocols　compromising　GPPD-labeled　HRP-anti-PRL,　nanogold　particles-labeled　HRP-anti-PRL　and　poly(o-phenylenediamine)　microspheres-labeled　HRP-anti-PRL,　were　investigated　for　detection　of　PRL,　and　improved　analytical　features　were　obtained　with　the　GPPD-based　strategy.　With　the　GPPD　labeling　method,　dependence　of　the　electrochemical　signals　on　the　incubation　time　and　pH　of　the　assay　solution　were　also　studied.　The　strong　attachment　of　HRP-anti-PRL　to　the　GPPDs　resulted　in　a　good　repeatability　and　intermediate　reproducibility　down　to　9.8%.　The　dynamic　concentration　range　spanned　from　0.5　to　180ngmL　-1　PRL　with　a　detection　limit　of　0.1ngmL　-1　at　the　3S　blank　level.　No　significant　differences　at　the　95%　confidence　level　were　encountered　in　the　analysis　of　10　spiked　blank　cattle　serum　samples　between　the　developed　immunoassay　and　enzyme-linked　immunosorbent　assay　method　for　determination　of　PRL.　©　2012　Elsevier　B.V.