Hyaluronidase　(HAase)　has　been　regarded　as　one　of　the　potential　cancer　biomarkers.　In　this　study,　a　novel　enzyme-responded　controlled　release　electrochemical　biosensor　has　been　constructed　for　HAase　detection.　Methylene　blue　(MB)　can　be　adsorbed　on　hyaluronic　acid　(HA)　to　form　macromolecular　HA–MB　complex　first　through　electrostatic　interaction　between　anionic　HA　and　positively　charged　MB.　The　prepared　complex　cannot　be　filtered　out　through　centrifugal　ultrafiltration,　and　small　electrochemical　signal　can　be　recorded　from　the　ultrafiltrate.　With　the　help　of　HAase,　HA–MB　complex　can　be　cleaved　into　small　pieces　through　enzyme　digestion.　The　small　pieces　of　HA–MB　complex　can　be　easily　released　from　the　macromolecular　HA–MB　complex　to　the　ultrafiltrate　by　centrifugal　ultrafiltration.　Then,　the　enhanced　electrochemical　signal　from　MB　can　be　detected　in　the　above-mentioned　ultrafiltrate.　The　enhanced　differential　pulse　voltammetric　response　of　the　system　has　a　linear　relationship　with　HAase　concentration　in　the　range　of　4.0–60　U·mL−1,　with　the　limit　of　detection　of　0.28　U·mL−1.　The　developed　strategy　has　been　successfully　applied　to　detect　HAase　activity　in　urine　samples.　©　2021,　The　Nonferrous　Metals　Society　of　China.