A　homogeneous　electrochemiluminescence　(ECL)　sensor　for　hyaluronidase　(HAase)　detection　was　developed　by　controlling　the　coating　of　hyaluronic　acid　(HA)　on　the　surface　of　magnetic　ECL　nanoparticles　(NPs).　Initially,　Fe3O4@Ru@SiO2　NPs　with　magnetic　properties　were　synthesized　and　subsequently　coated　with　HA　(Fe3O4@Ru@SiO2-HA).　The　properties　of　HA,　such　as　its　negative　charge　and　viscosity,　induced　strong　electrostatic　repulsion　and　reduced　electron　transfer　efficiency　between　the　Fe3O4@Ru@SiO2-HA　NPs　and　the　negatively　charged　electrodes.　Furthermore,　this　coating　hindered　the　approach　of　co-reactants　to　the　NPs,　resulting　in　a　diminished　ECL　signal.　In　the　presence　of　HAase,　HA　is　hydrolyzed,　releasing　the　Fe3O4@Ru@SiO2　NPs.　Magnetic　separation　minimizes　background　interference　and　allows　the　magnetic　ECL　NPs　to　diffuse　freely　in　solution,　facilitating　electron　transfer　and　reactant　interaction,　thereby　leading　to　detectable　signal　changes.　The　sensor　exhibited　a　linear　response　to　HAase　concentration　ranging　from　5.0　to　60　U/mL,　with　a　detection　limit　of　1.8　U/mL.　This　method　is　rapid,　straightforward,　and　effectively　leverages　the　inherent　properties　of　HA,　making　it　suitable　for　broad　applications　in　the　detection　of　proteins,　enzymes,　and　other　biomarkers.　©　2024　Elsevier　B.V.