A　novel　electrochemiluminescence　(ECL)　biosensor　for　hyaluronidase　(HAase)　based　on　the　adjustable　electrostatic　interaction　between　the　surface-charge-controllable　nanoparticles　and　negatively　charged　electrode　has　been　devised.　Hyaluronic　acid　(HA)-coated　amino-modified　ruthenium　bipyridine-doped　silica　nanoparticles　(Ru@SiO2-NH2@HA　NPs)　have　been　synthesized　and　act　as　ECL　indicators,　and　the　surface　of　this　particle　is　negatively　charged　because　HA　contains　a　large　amount　of　OH-　and　COO-.　The　strong　electrostatic　repulsion　between　the　Ru@SiO2-NH2@HA　NPs　and　negatively　charged　indium　tin　oxide　(ITO)　electrode　surface　leads　to　the　detection　of　a　low-intensity　ECL　signal.　In　the　presence　of　HAase,　the　HA　on　the　surface　of　the　Ru@SiO2-NH2@HA　NPs　can　be　decomposed,　and　the　particles　can　be　transformed　into　positively　charged　amino-modified　ruthenium　bipyridine-doped　silica　nanoparticles　(Ru@SiO2-NH2NPs),　which　can　be　concentrated　near　the　surface　of　the　ITO　electrode　through　electrostatic　attraction,　and　result　in　the　detection　of　an　enhanced　ECL　signal.　The　ECL　of　the　system　has　a　good　linear　relationship　with　HAase　concentration　in　the　range　of　2.0-60　U/mL,　and　the　limit　of　detection　was　0.37　U/mL.　The　designed　biosensor　had　been　applied　to　detect　the　target　in　real　samples　with　satisfied　results.　©　2022　American　Chemical　Society.　All　rights　reserved.