Vertically-ordered　mesoporous　silica　film　(VMSF),　with　large　amount　of　small　pores　and　abundant　negative　charges,　had　been　modified　on　the　surface　of　indium　tin　oxide　electrodes　and　applied　to　develop　an　electrochemiluminescence　(ECL)　biosensor　for　acute　myeloid　leukemia　biomarker　hsa-miR-10a-5p　(chosen　as　a　model　target).　The　small　ECL　indicator　(Ru(phen)32+)　can　be　enriched　in　the　pores　through　electrostatic　adsorption　and　then　diffuse　to　the　electrode　surface　to　produce　strong　ECL　signals.　The　presence　of　target　can　trigger　hyperbranched　rolling　circle　amplification　(HRCA)　and　produce　a　large　amount　of　double-stranded　DNA　(dsDNA).　Ru(phen)32+　can　embed　into　the　groove　structure　of　dsDNA　and　forms　negatively　charged　large-sized　complexes,　which　cannot　pass　through　the　narrow　pores　of　VMSF　to　reach　the　electrode.　This　led　to　a　significant　decrease　of　ECL　signal　of　the　system.　The　change　of　the　ECL　intensity　of　the　system　had　a　linear　relationship　with　logarithm　of　target　concentration　within　the　range　of　1　fM–1　nM.　The　limit　of　detection　was　1.23　fM.　The　developed　biosensor　had　been　applied　to　determine　the　target　substance　in　serum　samples　with　satisfied　results　without　complicated　sample　pretreatment.　The　introduction　of　VMSF　endows　the　biosensor　with　superior　anti-interference　capability　and　simpler　operational　steps,　promising　applications　in　the　diagnosis　of　acute　myeloid　leukemia　and　potential　extension　to　the　analysis　of　other　target　molecules　through　primer　design.　©　2024　Elsevier　B.V.