Free　copper　ions　(Cu+　and　Cu2+)　have　critical　toxicity　to　cells,　although　copper　is　an　essential　element　for　human　body.　Hence,　sensitive　monitoring　is　crucial　to　avoid　over　intake　of　Cu+/Cu2+.　We　herein　designed　a　ssDNA　sequence　(A31)　for　synthetizing　A31-templated　silver　nanoclusters　(AgNCs),　and　demonstrated　that　Cu+/Cu2+　can　induce　the　aggregation　of　A31-templated　AgNCs　and　thus　greatly　enhanced　the　fluorescence　emission　of　A31-templated　AgNCs.　Based　on　Cu+/Cu2+-induced　fluorescence　enhancement　effect　of　A31-templated　AgNCs,　a　label-free　and　signal-on　fluorescent　sensing　platform　was　developed　for　the　specific　and　sensitive　detection　of　Cu+/Cu2+　in　biological　samples　and　intracellular　imaging　of　Cu+/Cu2+　in　cells.　The　signal-on　fluorescent　sensing　platform　could　be　used　to　rapidly　detect　Cu+　and　Cu2+　with　a　detection　limit　of　0.1　µM　within　30　min.,　and　to　perform　the　intracellular　imaging　of　Cu+　and　Cu2+　in　cells　with　good　cell　permeability　and　biocompatibility.　By　using　the　signal-on　fluorescent　sensing　platform,　we　have　successfully　detected　Cu+　and　Cu2+　in　cells　fluids　and　human　serum　with　a　recovery　of　90–104%　and　a　RSD　(n　=　5)　+/Cu2+　in　Hela　cells.　The　developed　fluorescent　sensing　platform　has　obvious　analytical　and　imaging　advantages　such　as　signal-on,　simple　operation,　short　analysis　time,　both　Cu+　and　Cu2+　detection,　similar　or　higher　sensitivity,　good　cell　permeability　and　biocompatibility,　which　promising　a　reliable　approach　for　the　rapid　and　on-site　detection　or　imaging　of　free　copper　ions　in　biological　samples　in　clinical　diagnosis.　©　2022　Elsevier　B.V.