The　development　of　simple　and　sensitive　method　for　hyaluronidase　(HAase)　detection　is　significant　for　the　clinical　diagnosis　and　treatment　of　cancer　as　HAase　is　a　potential　tumor　marker.　In　this　study,　a　simple　but　sensitive　electrochemiluminescence　(ECL)　biosensor　has　been　designed　for　HAase　detection　based　on　the　electrostatic　interaction　of　anionic　hyaluronic　acid　(HA)　and　cationic　luminous　reagent　Ru(bpy)32+.　Ru(bpy)32+　is　adsorbed　on　HA　through　electrostatic　interaction　to　form　HA-Ru(bpy)32+　complex.　The　Ru(bpy)32+　in　the　HA-Ru(bpy)32+　complex　cannot　be　filtered　out　because　of　its　large　molecular　weight.　In　the　presence　of　HAase,　the　HA　in　HA-Ru(bpy)32+　can　be　cleaved　into　fragments　by　enzymatic　hydrolysis.　As　a　result,　the　Ru(bpy)32+　attached　on　the　HA　fragments　can　be　separated　from　the　HA-Ru(bpy)32+　complex　by　centrifugation　easily.　The　resulting　ultrafiltrate　containing　Ru(bpy)32+　can　be　used　to　characterize　the　concentration　of　HAase　in　the　sample.　The　ECL　intensity　of　Ru(bpy)32+　has　a　linear　relationship　with　the　concentration　of　HAase　in　the　range　of　2.0–40　U/mL　with　a　detection　limit　of　0.33　U/mL.　The　proposed　ECL　system　has　been　applied　to　detect　HAase　in　urine　samples　with　satisfied　results　and　HAase　inhibitor　with　high　efficiency.　©　2018　Elsevier　B.V.