Detecting　of　hyaluronidase　(HAase)　activity　is　vital　in　oncology　as　it　drives　tumor　invasion　and　metastasis　by　degrading　hyaluronic　acid　(HA)　in　the　extracellular　matrix.　In　this　study,　combining　a　target-responsive　hydrogel　with　magnetic　nanozymes,　a　novel　colorimetric　sensor　was　designed　for　HAase　quantification.　The　composite　of　Fe₃O₄　and　MoS₂　(Fe3O4@MoS2)　has　magnetism　activity　and　strong　peroxidase-like　activity,　simultaneously　has　embedded　in　a　hydrogel　matrix　format　from　HA,　the　presence　of　HAase　can　specific　hydrolysis　HA,　and　caused　the　rupture　of　the　hydrogel　and　results　in　the　releasing　of　the　Fe3O4@MoS2.　The　released　Fe3O4@MoS2　can　be　collected　through　magnetic　separation　easily　and　then　applied　to　catalyze　the　oxidation　of　3,3′,5,5′-tetramethylbenzidine　(TMB)　by　H2O2,　producing　a　distinct　color　change,　which　can　be　detected　by　naked　eyes　easily.　The　absorbance　of　the　system　had　a　good　linear　response　with　HAase　concentration　within　the　range　of　2.5–90　U/mL,　with　a　limit　of　detection　(LOD)　of　1.4　U/mL　(S/N　=　3).　The　proposed　sensor　was　successfully　applied　to　detect　HAase　in　serum　samples　from　healthy　individuals　and　pancreatic　cancer　patients,　yielding　satisfactory　results.　©　2025　Elsevier　B.V.