Lead　(Pb2　+)　contamination　poses　significant　risks　to　human　health　and　the　environment,　necessitating　the　development　of　sensitive　and　rapid　detection　methods.　In　this　study,　we　present　a　DNA　nanosphere-enhanced　substrate　strand-DNAzyme　(DS-Sub-Dz)　system　for　the　efficient　detection　of　Pb2+　in　both　environmental　and　biological　samples.　The　DS　was　constructed　by　annealing　single-stranded　DNA　with　four　palindromic　regions　and　extending　sequences　at　the　terminals,　simplifying　the　process　and　reducing　costs　compared　to　traditional　long　DNA　strands.　The　Sub-Dz,　modified　with　fluorophore　(FAM)　and　quencher　(BHQ1),　was　then　grafted　onto　the　DS　surface,　forming　the　DS-Sub-Dz　system　through　a　hybridization　reaction.　This　system　utilizes　spatial　confinement　and　a　cleavage　recycling　reaction　to　amplify　the　fluorescence　signals　of　FAM,　enabling　highly　sensitive　and　rapid　detection　of　Pb2+　over　a　range　of　2　nM　to　40　μM.　With　a　detection　limit　(LOD)　of　2.0　nM-1000　times　lower　than　the　control　Sub-Dz　system-the　DS-Sub-Dz　demonstrated　exceptional　sensitivity.　Further　tests　confirmed　its　ability　to　distinguish　between　various　heavy　metal　ions　in　tap　water.　The　enhanced　cell　internalization　efficiency,　accelerated　reaction　kinetics,　and　improved　biostability　of　the　DS-Sub-Dz　system　also　enabled　successful　Pb2+　detection　in　both　water　samples　and　intracellular　imaging,　showcasing　its　potential　for　monitoring　lead　contamination　in　environmental　and　body　safety　applications.　©　2025　Elsevier　B.V.