The　sensing　performance　of　a　microchannel-based　electrochemiluminescence　(ECL)　biosensor　is　related　to　the　change　ratio　of　charge　density　on　the　surface　of　microchannels　caused　by　a　target　recognition　reaction.　In　this　study,　adenosine　triphosphate　(ATP)　served　as　a　model　target.　The　dsDNA　superstructures　containing　a　capture　probe　(CP,　containing　an　ATP　aptamer　sequence)　and　alternating　units　of　ssDNA　probes　of　P1　and　P2,　CP/(P1/P2)n,　were　grafted　onto　the　inner　wall　of　microchannels　first.　The　CP　in　dsDNA　superstructures　captured　ATP　molecules,　causing　the　release　of　dsDNA　fragments　containing　alternating　units　of　P1　and　P2,　(P1/P2)n.　The　target　recognition　reaction　significantly　changed　the　charge　density　of　microchannels,　which　altered　the　ECL　intensity　of　the　(1,10-phenanthroline)ruthenium(II)/tripropylamine　system　in　the　reporting　interface.　The　ECL　intensity　of　the　constructed　system　had　a　linear　relationship　with　the　logarithm　of　ATP　concentration　ranging　from　1　fM　to　100　pM　with　a　detection　limit　of　0.32　fM　(S/N　=　3).　The　biosensor　was　successfully　applied　to　detect　ATP　in　rat　brains.　©　2022　American　Chemical　Society.