Herein,　we　present　an　innovative　electrochemiluminescence　(ECL)　biosensor　for　the　ultrasensitive　detection　of　N-nitrosodimethylamine　(NDMA).　The　biosensor　utilizes　a　triple　signal　amplification　strategy,　combining　rolling　circle　amplification　(RCA),　CRISPR/Cas12a-driven　hyperbranched　rolling　circle　amplification　(HRCA),　and　electrostatic　repulsion　with　size　exclusion　effects　from　vertically　ordered　mesoporous　silica　film　(VMSF)/indium　tin　oxide　(ITO)　on　double-stranded　DNA　(dsDNA)-Ru(phen)32+　complexes.　In　this　system,　aptamers　and　circular　DNA　undergo　RCA　reactions,　followed　by　the　CRISPR/Cas12a-mediated　HRCA　process,　producing　abundant　dsDNA.　The　electropositive　ECL　indicator,　namely　Ru(phen)32+,　was　subsequently　adsorbed　onto　the　electronegative　dsDNA,　forming　dsDNA-Ru(phen)32+　complexes.　These　complexes　are　subjected　to　electrostatic　repulsion　and　size　exclusion　by　the　VMSF-modified　ITO　electrode,　resulting　in　a　lower　ECL　intensity.　Upon　introducing　NDMA,　the　aptamer　preferentially　binds　to　NDMA,　thereby　preventing　the　formation　of　long　dsDNA.　This　process　releases　free　Ru(phen)32+,　which　diffuses　to　the　electrode　surface　through　narrow　mesoporous　channels　via　electrostatic　adsorption.　Consequently,　an　enhanced　and　strong　ECL　signal　is　observed.　The　integration　of　VMSF　enhances　selectivity　and　sensitivity　by　excluding　larger　impurities　and　promoting　the　electrostatic　repulsion　of　dsDNA-Ru(phen)32+　complexes　near　the　electrode　surface.　Additionally,　the　CRISPR/Cas12a　system　eliminates　the　formation　of　primer　dimers　and　reduces　false　positives　through　its　unique　cis-　and　trans-cleavage　activities.　The　biosensor　demonstrated　excellent　performance　with　a　linear　correlation　between　the　ECL　signal　and　NDMA　concentration　in　the　range　spanning　from　10　pg/mL　to　10　μg/mL,　achieving　a　low　limit　of　detection　of　5.33　pg/mL.　This　platform　offers　a　reliable　and　robust　solution　for　detecting　NDMA　in　complex　matrices,　making　it　a　promising　tool　for　environmental　monitoring,　public　health,　and　safety　applications.　©　2025　American　Chemical　Society.