A　cell-free　RNA　transcription　system　had　been　coupled　with　electrochemiluminescence　(ECL)　detection　technology　for　the　first　time　to　develop　an　ascorbic　acid　(AA,　acting　as　a　model　target)　biosensor.　The　biosensor　is　composed　of　single-stranded　DNA　(ssDNA)　sequences　modified　with　alkynyl　and　azido　groups,　respectively,　alongside　an　incomplete　gene　circuit　framework.　The　addition　of　target　AA　and　copper　ions　will　cause　the　linkage　of　the　two　ssDNA　sequences　through　a　click　chemistry　reaction.　This　results　in　the　subsequent　reconstruction　of　a　complete　gene　circuit.　The　reconstituted　gene　circuit,　in　conjunction　with　the　T7　RNA　polymerase,　drives　the　transcription　of　substantial　quantities　of　RNA.　ssDNA　labeled　with　ferrocene　(Fc)　(Fc-DNA)　had　been　immobilized　on　a　tris(2,2′-bipyridyl)　ruthenium(II)　chloride　hexahydrate-doped　SiO2　nanoparticle　(Ru@SiO2　NPs)　modified　electrode　first.　The　quenching　effect　of　Fc　on　Ru@SiO2　causes　the　low　ECL　detected.　The　transcribed　RNA　sequence　assisted　double-stranded　specific　nuclease　(DSN)　to　cut　the　ssDNA-Fc　and　the　ECL　of　the　system　was　enhanced.　Optimal　experimental　conditions　reveal　that　the　ECL　signal　exhibits　a　linear　correlation　with　the　logarithmic　concentration　of　AA,　spanning　a　detection　range　from　100　nM　to　1　mM,　with　a　detection　limit　of　45　nM.　This　innovative　methodology　expands　the　utility　of　a　cell-free　RNA　transcription　system　within　the　realm　of　biosensing　applications.　©　2024　American　Chemical　Society.