A　highly　sensitive　and　selective　fluorescence　biosensor　for　inorganic　pyrophosphatase　(PPase)　activity　has　been　developed　based　on　special　click　ligation　trigger　hyperbranched　rolling　circle　amplification　(CLT-HRCA).　Pyrophosphate　ion　(PPi)　can　coordinate　with　Cu　2+　to　form　stable　PPi/Cu　2+　complex　and　Cu　2+　in　the　complex　cannot　be　reduced　to　Cu　+　.　The　addition　of　PPase　causes　the　hydrolysis　of　PPi　into　orthophosphate　(Pi)　and　therefore　induces　the　releasing　of　Cu　2+　from　the　stable　PPi/Cu　2+　complex,　and　the　free　Cu　2+　is　easily　reduced　to　Cu　+　by　sodium　ascorbate.　Then　Cu　+　catalyzes　the　cyclization　reaction　between　the　specially　designed　5′-azide　and　3′-alkyne　tagged　padlock　probes　through　Cu　+　catalyzed　azide-alkyne　cycloaddition　(CuAAC),　which　in　turn　initiates　the　hyperbranched　rolling　circle　amplification　(HRCA).　Given　that　the　CLT-HRCA　products　contain　large　amounts　of　double-stranded　DNAs　(dsDNAs),　the　addition　of　SYBR　Green　I　resulted　in　the　enhanced　fluorescence　signal.　There　was　a　linear　relationship　between　the　enhanced　fluorescence　intensity　and　the　logarithm　PPase　activity　ranging　from　0.05　to　25 mU　with　a　detection　limit　of　0.02 mU.　Such　proposed　biosensor　has　been　successfully　applied　to　screen　the　potential　PPase　inhibitors　and　has　accessed　the　related　inhibit　ability　with　high　efficiency.　©　2016,　Springer-Verlag　Berlin　Heidelberg.