Hirschsprung’s　disease　(HSCR),　a　congenital　condition　characterized　by　the　absence　of　nerve　cells　in　the　intestinal　wall,　often　requires　early　and　accurate　diagnosis　for　optimal　patient　outcomes.　In　this　study,　we　developed　a　novel　and　ultrasensitive　biosensing　strategy　for　the　detection　of　HSCR-related　microRNAs　(miRNAs)　by　integrating　catalytic　hairpin　assembly　(CHA)　with　CRISPR-Cas12a　technology.　A　two-stage　process　consists　of　array　recognition,　and　a　universal　readout　is　designed.　In　the　first　stage,　target　miRNAs　are　recognized　and　amplified　on　a　solid-phase　microarray,　while　in　the　second　stage,　the　accumulated　conversion　chains　which　are　not　related　to　target　sequences,　activate　Cas12a,　leading　to　the　cleavage　of　reporter　DNA　and　the　generation　of　a　fluorescence　signal　spatially　separated　from　the　first　stage.　The　proposed　method　was　validated　for　the　comprehensive　detection　of　HSCR-related　miRNAs　and　demonstrated　high　sensitivity　and　specificity.　This　work　represents　a　significant　advancement　in　miRNA　diagnostics　and　holds　potential　for　broader　clinical　applications.　©　The　Author(s),　under　exclusive　licence　to　Springer-Verlag　GmbH　Austria,　part　of　Springer　Nature　2025.