Electrochemiluminescence　(ECL)　arises　from　the　collisional　annihilation　between　the　electro-excited　luminophore　and　co-reactant.　Most　ECL-based　bioassays　require　extensive　free　co-reactants　that　collide　with　luminophores　randomly.　Herein,　the　N,N-dimethylaniline　(DMA)　group　was　electro-grafted　on　the　ITO　electrode　surface　as　the　bottom　co-reactant　layer,　and　the　vertically　ordered　mesoporous　silica　film　(VMSF)　was　prepared　as　the　upper　layer　to　regulate　the　accessibility　of　Ru(bpy)32+.　Thus,　the　dual-layer　functionalized　VMSF/N-ITO　was　newly　built　for　the　controllable　ECL　study.　Relevant　conditions,　including　the　DMA　electro-grafting,　VMSF　thickness,　buffer　concentration　and　pH　value,　were　discussed　to　improve　the　ECL　efficiency.　Based　on　the　VMSF/N-ITO　platform,　a　homogeneous　ECL　bioassay　was　developed　for　miRNA-21　detection.　The　target　nucleotide　sequence　induced　the　hybridization　chain　reaction　(HCR)　of　probes　H1　and　H2　to　absorb　free　Ru(bpy)32+　as　much　as　possible.　Residual　Ru(bpy)32+　permeated　the　VMSF　nanochannels　to　collide　with　the　DMA　co-reactant　in　producing　an　ECL　response.　There　was　a　linear　relationship　between　∆ECL　intensity　and　logarithmic　concentration　of　miRNA-21　in　the　1　fM　∼　100　pM　range,　and　the　detection　limit　was　0.25　fM.　The　developed　approach　was　probes　immobilization-free,　highly　sensitive,　and　anti-interference,　opening　a　new　avenue　for　ECL-based　bioassays.　©　2023　Elsevier　B.V.