Homogeneous　electrochemiluminescence　(ECL)　has　gained　attention　for　its　simplicity　and　stability.　However,　false　positives　due　to　solution　background　interference　pose　a　challenge.　To　address　this,　magnetic　ECL　nanoparticles　(Fe3O4@Ru@SiO2　NPs)　were　synthesized,　offering　easy　modification,　magnetic　separation,　and　stable　luminescence.　These　were　utilized　in　an　ECL　sensor　for　miRNA-155　(miR-155)　detection,　with　locked　DNAzyme　and　substrate　chain　(mDNA)　modified　on　their　surface.　The　poor　conductivity　of　long-chain　DNA　significantly　impacts　the　conductivity　and　electron　transfer　capability　of　Fe3O4@Ru@SiO2　NPs,　resulting　in　weaker　ECL　signals.　Upon　target　presence,　unlocked　DNAzyme　catalyzes　mDNA　cleavage,　leading　to　shortened　DNA　chains　and　reduced　density.　In　contrast,　the　presence　of　short-chain　DNA　has　minimal　impact　on　the　conductivity　and　electron　transfer　capability　of　Fe3O4@Ru@SiO2　NPs.　Simultaneously,　the　material　surface’s　electronegativity　decreases,　weakening　the　electrostatic　repulsion　with　the　negatively　charged　electrode,　resulting　in　the　system　detecting　stronger　ECL　signals.　This　sensor　enables　homogeneous　ECL　detection　while　mitigating　solution　background　interference　through　magnetic　separation.　Within　a　range　of　100　fM　to　10　nM,　the　sensor　exhibits　a　linear　relationship　between　ECL　intensity　and　target　concentration,　with　a　26.91　fM　detection　limit.　It　demonstrates　high　accuracy　in　clinical　sample　detection,　holding　significant　potential　for　clinical　diagnostics.　Future　integration　with　innovative　detection　strategies　may　further　enhance　sensitivity　and　specificity　in　biosensing　applications.　©　2024　American　Chemical　Society