The　abnormal　expression　of　pyrophosphatase　(PPase)　is　closely　related　to　many　diseases　and　malignant　tumors,　so　the　detection　for　PPase　is　of　great　significance　in　clinical　diagnosis,　disease　monitoring,　and　other　biomedical　aspects.　In　this　study,　a　sensitive　and　specific　electrochemiluminescence　(ECL)　biosensor　combined　highly　specific　Cu+-catalyzed　azide-alkyne　cycloaddition　(CuAAC)　with　high　efficiency　of　hybridization　chain　reaction　(HCR)　for　the　purpose　of　detecting　pyrophosphatase　has　been　designed.　Highly　efficient　hybridization　chain　reaction　amplification　processed　in　homogeneous　solution　and　the　amplification　products　were　connected　to　the　electrode　surface　in　one　step,　which　solved　the　problem　of　low　DNA　amplification　efficiency　on　the　electrode　surface　because　of　the　steric　hindrance.　Ru(phen)32+　was　embedded　into　the　dsDNA　and　functioned　as　ECL　probes;　the　enhanced　ECL　intensity　of　the　system　had　a　linear　relationship　with　the　logarithm　of　PPase　concentration　in　the　range　of　0.025-50　mU　with　a　detection　limit　of　8　μU.　The　method　was　proved　to　be　of　good　specificity,　repeatability,　and　stability　that　could　be　used　for　screening　and　quantitatively　determining　pyrophosphatase　inhibitor　sodium　fluoride.　The　practicability　of　this　method　in　clinical　application　has　been　proved　through　the　detection　of　serum　from　the　clinical　arthritis　patients.　Moreover,　the　method　can　be　used　to　monitor　PPase　activity　of　arthritis　patients　before　and　after　administration　to　provide　reference　for　the　effect　of　drug　treatment.　Copyright　©　2020　American　Chemical　Society.