Hyaluronidase　(HAase),　a　specific　enzyme　of　hyaluronic　acid　(HA),　has　been　reported　as　a　potential　tumor　biomarker　in　recent　years.　Hence,　developing　some　simple,　rapid,　and　sensitive　methods　for　HAase　assay　is　necessary.　In　this　work,　a　simple　and　sensitive　biosensor　constructed　by　a　reliable　controlled　release　system　and　a　mature　electrochemiluminescence　(ECL)　analytical　technique　has　been　devised　for　the　quantification　of　HAase　with　high　efficiency　and　selectivity.　Tris　(2,2′-bipyridyl)　ruthenium(II)　chloride　hexahydrate　doped　SiO2　nanoparticles　(Ru@SiO2　NPs),　as　ECL　signal　probes,　were　trapped　in　the　hydrogel　fabricated　by　HA　and　polyethylenimine　evenly　and　steadily.　When　HAase　existed,　the　hydrogel　was　decomposed　by　HAase,　and　the　Ru@SiO2　NPs　escaped　from　the　hydrogel　into　the　supernate.　The　ECL　signal　produced　from　the　supernate　can　be　detected　and　used　to　characterize　HAase　concentration.　The　result　showed　a　good　linear　relationship　between　ECL　intensity,　and　HAase　concentration　ranged　from　2　to　60　U/mL　and　the　limit　of　detection　was　2　U/mL.　The　developed　controlled　release　ECL　biosensor　has　been　used　for　HAase　quantification　in　urine　samples　with　satisfactory　results.　©　2019　American　Chemical　Society.