Sensitive　and　selective　detection　of　N-nitrosodimethylamine　(NDMA),　a　highly　carcinogenic　environmental　contaminant,　is　essential　for　public　health　and　safety.　In　this　study,　we　have　developed　a　homogeneous　electrochemiluminescence　(ECL)　biosensor　for　ultrasensitive　NDMA　detection　by　integrating　Nafion-functionalized　covalent　organic　framework　(COF)　with　aptamer　recognition　and　magnetic　separation.　The　imine-linked　COF　(COF-LZU1)　scaffold　not　only　enables　stable　encapsulation　of　Ru(bpy)32　+,　but　also　creates　a　confined　microporous　environment　that　locally　concentrates　c　(TPrA)　and　enhances　the　generation　and　survival　of　TPrA　radicals,　thereby　amplifying　the　ECL　signal.　Nafion　further　improves　Ru　loading　and　ionic　conductivity.　Biotinylated　aptamers　were　initially　immobilized　on　streptavidin-coated　magnetic　beads　(MBs)　and　hybridized　with　complementary　DNA-modified　Ru@COF-LZU1　to　form　Ru@COF-LZU1-MBs　complex.　Upon　NDMA　binding,　a　conformational　switch　releases　Ru@COF-LZU1　into　the　solution.　After　magnetic　separation,　the　ECL　signal　of　the　supernatant　is　measured　in　the　presence　of　TPrA.　This　homogeneous　format　avoids　electrode　surface　modification,　which　is　commonly　required　in　conventional　Ru@COF-LZU1-based　ECL　sensors,　offering　improved　reproducibility　and　simplified　operation.　The　biosensor　exhibits　a　wide　linear　range　(100　fg/mL　-　100　ng/mL)　with　a　low　detection　limit　of　9.11　fg/mL.　This　platform　offers　a　sensitive,　reproducible,　and　facile　strategy　for　monitoring　NDMA　and　similar　small-molecule　contaminants　in　complex　matrices.　©　2025　Elsevier　B.V.