A　highly　sensitive　＂two-site＂　chemiluminescent　immunoassay　specific　for　human　thyroid　stimulating　hormone　(TSH)　was　developed.　The　signal　amplification　was　achieved　via　a　biotin-streptavidin　system　(BSAS).　The　HRP-luminol-H2O2　chemiluminescent　system　with　high　sensitivity　was　chosen　as　the　detection　system.　Biotinylated　anti-TSH　monoclonal　antibody　(MAb)　and　HRP-labeled　streptavidin　were　first　synthesized.　Then　the　signal　amplification　was　achieved　through　the　interaction　between　the　biotinylated　anti-TSH　MAb　and　the　HRP-streptavidin　conjugate.　The　light　intensity　developed　was　in　proportion　to　the　TSH　present　in　the　samples.　The　assay　showed　little　cross-reactivity　with　three　other　glycoprotein　hormones　(human　chorionic　gonadotropin　(HCG),　luteinizing　hormone　(LH),　follicle　stimulating　hormone　(FSH))　due　to　the　high　specificity　of　the　antibody.　The　working　range　for　human　thyroid　stimulating　hormone　was　0.1-40　mU　L-1.　Both　the　intra-assay　and　inter-assay　coefficients　of　variation　were　less　than　10%　for　the　BSAS　based　chemiluminescent　enzyme　immunoassay　(CLEIA).　The　proposed　assay　had　a　sensitivity　of　0.01　mU　L-1　which　was　10-fold　higher　than　the　HRP-MAb　conjugate　based　TSH　immunoassay.　Thus　the　higher　sensitivity　facilitated　the　clinical　testing　for　thyroid　states.　The　effects　of　several　reaction　parameters,　such　as　incubation　time,　temperature,　and　reaction　volume　of　the　method,　were　also　studied.　This　method　has　been　successfully　applied　to　the　evaluation　of　TSH　in　human　serum.　Compared　with　the　commercial　enzyme　chemiluminescent　immunoassay,　the　correlation　was　satisfied.　©　2008　Elsevier　B.V.　All　rights　reserved.