A　novel　homogeneous　immunoassay　protocol　was　designed　for　quantitative　monitoring　of　small　molecular　biotoxin　(brevetoxin　B,　PbTx-2,　as　a　model)　by　using　target-responsive　cargo　release　from　polystyrene　microsphere-gated　mesoporous　silica　nanocontainer　(MSN).　Initially,　monoclonal　mouse　anti-PbTx-2　capture　antibody　was　covalently　conjugated　onto　the　surface　of　MSN　(mAb-MSN),　and　the　electroactive　cargo　(methylene　blue,　MB)　was　then　trapped　in　the　pores　of　mAb-MSN　by　using　aminated　polystyrene　microspheres　(APSM)　based　on　the　electrostatic　interaction.　Upon　addition　of　target　PbTx-2,　the　positively　charged　APSM　was　displaced　from　the　negatively　charged　mAb-MSN　because　of　the　specific　antigen-antibody　reaction.　Thereafter,　the　molecular　gate　was　opened,　and　the　trapped　methylene　blue　was　released　from　the　pores.　The　released　methylene　blue　could　be　monitored　by　using　a　square　wave　voltammetry　(SWV)　in　a　homemade　microelectrochemical　detection　cell.　Under　optimal　conditions,　the　SWV　peak　current　increased　with　the　increasing　of　PbTx-2　concentration　in　the　range　from　0.01　to　3.5　ng　mL(-1)　with　a　detection　limit　(LOD)　of　6　pg　mL(-1)　PbTx-2　at　the　3S(blank)　criterion.　Intra-　and　interassay　coefficients　of　variation　with　identical　batches　were　＜=　6%　and　9.5%,　respectively.　The　specificity　and　sample　matrix　interfering　effects　were　acceptable.　The　analysis　in　12　spiked　seafood　samples　showed　good　accordance　between　results　obtained　by　the　developed　immunoassay　and　a　commercialized　enzyme-linked　immunosorbent　assay　(ELISA)　method.　Importantly,　the　target-responsive　controlled　release　system-based　electrochemical　immunoassay　(CRECIA)　offers　a　promising　scheme　for　the　development　of　advanced　homogeneous　immunoassay　without　the　sample　separation　and　washing　procedure.