A　novel　signal-amplified　strategy　for　sensitive　electrochemical　immunoassay　of　cancer　marker　(human　tissue　polypeptide　antigen,　TPA,　used　in　this　case)　is　developed　by　using　Prussian　blue　nanoparticles-doped　nanogold　microsphere　(AuPB)　as　the　promoter.　To　construct　such　an　immunoassay,　the　AuPB　was　initially　synthesized　by　using　the　reverse　micelle　method,　and　the　as-synthesized　AuPB　was　then　heavily　functionalized　with　horseradish　peroxidase　(HRP)　and　anti-TPA　antibody.　Based　on　a　specific　sandwich-type　immunoassay　format,　target　TPA　was　monitored　on　anti-TPA-functionalized　glassy　carbon　electrode　by　using　the　biofunctional　AuPB　as　the　signal　tag.　Compared　with　conventional　nanogold　labeling,　the　as-prepared　AuPB　possessed　good　redox　activity,　which　could　be　employed　as　an　electron　mediator　for　improvement　of　catalytic　efficiency　of　the　labeled　HRP.　Under　optimal　conditions,　the　electrochemical　immunoassay　presents　good　electrochemical　responses　toward　target　TPA,　and　allowed　the　detection　of　TPA　at　a　concentration　as　low　as　5　pg　mL(-1).　The　precision,　reproducibility,　specificity　and　stability　of　the　electrochemical　immunoassay　are　acceptable.　In　addition,　the　methodology　is　validated　for　the　analysis　of　8　clinic　human　serum　specimens　and　8　spiked　serum　samples,　receiving　in　good　accordance　with　the　results　obtained　from　the　referenced　enzyme-linked　immunosorbent　assay　(ELISA)　method.