This　study　developed　an　ultrasensitive　method　involving　(1)　gold　nanoparticles　encoded　with　a　double-stranded　DNA　as　the　first　signal　amplification　and　goat　anti-staphylococcal　enterotoxin　B　(SEB)　polyclonal　antibody　and　(2)　magnetic　microparticles　coated　with　anti-SEB　monoclonal　antibody　to　detect　SEB.　Both　functionalized　nanoparticles　can　capture　SEB　in　a　sandwich　system.　The　released　DNA　barcodes　were　then　characterized　through　SYBR　Green　real-time　polymerase　chain　reaction　and　resulted　in　the　second　signal　amplification.　Under　optimized　experimental　conditions,　an　ultrasensitive　bio-barcode　for　SEB　detection　was　established,　and　its　detection　limit　could　reach　0.269　pg　mL(-1),　which　was　1000-fold　lower　than　that　of　conventional　enzyme-linked/immunosorbent　assay.　The　proposed　method　shows　great　potential　in　food　security.