Elevated　hyaluronidase　(HAase)　concentration　has　been　found　in　the　urine　samples　of　the　patients　with　bladder　cancer　and　development　of　simple　but　sensitive　detection　methods　for　HAase　is　important　for　related　disease　screening.　Pregnancy　test　strips　(PTSs)　is　one　of　the　most　common　portable　and　inexpensive　household　testing　strips,　which　has　been　used　to　measure　human　chorionic　gonadotropin　(HCG)　in　urine　sample　for　pregnancy　test.　In　this　study,　a　portable　biosensor　has　been　developed　for　HAase　using　PTSs　as　detection　device.　HCG　was　embedded　in　a　hydrogel　format　through　the　cross-linking　of　hyaluronic　acid　(HA)　and　polyethylenimine　(PEI).　The　presence　of　HAase　can　digest　HA　in　the　hydrogel　and　results　in　the　releasing　of　HCG,　which　can　be　detected　by　a　PTS　easily.　The　color　changing　of　the　test　line　(T　line)　indicates　the　existence　of　the　target　object　and　the　depth　of　color　can　be　used　for　semi-quantitative　detection　of　the　target　concentration.　The　ImageJ　software　was　used　to　extract　the　RGB　value　of　the　color　of　T　line.　The　different　of　the　RGB　value　(ΔRGB)　at　present　and　absent　of　target　has　a　strong　linear　correlation　with　the　HAase　concentration　between　1　U/mL　and　100　U/mL,　with　a　limit　of　detection　of　0.27　U/mL　(S/N　=　3).　The　proposed　method　was　used　to　detect　HAase　in　urine　samples　from　non-pregnancy　healthy　individuals　and　patients　diagnosed　with　bladder　cancer　as　a　demonstration.　©　2025　Elsevier　B.V.