Single　stranded　DNA　often　forms　stable　secondary　structures　under　physiological　conditions.　These　DNA　secondary　structures　play　important　physiological　roles.　However,　the　analysis　of　such　secondary　structure　folded　DNA　is　often　complicated　because　of　its　high　thermodynamic　stability　and　slow　hybridization　kinetics.　In　this　article,　we　demonstrate　that　Y-shaped　junction　probes　could　be　used　for　rapid　and　highly　efficient　detection　of　secondary　structure　folded　DNA.　Our　approach　contained　a　molecular　beacon　(MB)　probe　and　an　assistant　probe.　In　the　absence　of　target,　the　MB　probe　failed　to　hybridize　with　the　assistant　probe.　Whereas,　the　MB　probe　and　the　assistant　probe　could　cooperatively　unwind　the　secondary　structure　folded　DNA　target　to　form　a　ternary　Y-shaped　junction　structure.　In　this　condition,　the　MB　probe　was　also　opened,　resulting　in　separating　the　fluorophores　from　the　quenching　moiety　and　emitting　the　fluorescence　signal.　This　approach　allowed　for　the　highly　sensitive　detection　of　secondary　structure　folded　DNA　target,　such　as　a　tau　specific　DNA　fragment　related　to　Alzheimer＇s　disease　in　this　case.　Additionally,　this　approach　showed　strong　SNPs　identifying　capability.　Furthermore,　it　was　noteworthy　that　this　newly　proposed　approach　was　capable　of　detecting　secondary　structure　folded　DNA　target　in　cell　lysate　samples.　©　2012　Elsevier　B.V.