Electrochemiluminescence　(ECL)　has　achieved　significant　commercial　success　over　the　past　few　decades　across　various　fields,　particularly　in　the　healthcare　industry.　The　measurement　scheme　oftentimes　involves　target　recognition　elements　(e.g.　catching　antibodies)　labeled　with　a　suitable　ECL　luminophore　(e.g.　tris(2,2′-bipyridine)ruthenium(II))).　While　this　approach　realizes　the　ultrasensitive　detection　of　various　biomarkers,　it　is　somewhat　complicated　strategy　for　certain　targets　such　as　enzymes.　In　this　study,　β-glucuronidase　(B-GLU),　a　promising　biomarker　and　a　common　water/foodstuff　safety　indicator,　was　quantified　by　measuring　the　ECL　signal　of　fluorescent　product　generated　from　non-fluorescent　substrate　by　the　B-GLU　enzyme.　To　this　end,　hot　electron-induced　ECL　of　three　luminophores　(fluorescein,　4-methylumbelliferyl　and　resorufin)　that　are　used　as　building　blocks　to　synthesize　various　commercially　available　non-fluorescent　substrates　was　compared　for　the　first　time.　To　increase　the　appeal　and　practicality　of　this　approach,　the　common　multi-well　assay　format　was　adapted　to　the　present　type　ECL　by　carrying　out　the　ECL　reactions　at　single　carbon　black/polystyrene　electrode.　In　this　electrochemical　setup,　multiple　cells　were　fabricated　on　the　surface　of　a　poorly　conducting　substrate　by　attaching　Teflon　tape　with　multiple　holes　to　the　substrates　surface.　Sample　throughput　time　decreases　considerable　as　target,　blank　and　sample　signals　can　be　simultaneously　obtained　from　the　electrochemical　cells　when　voltage　is　applied　across　the　single　electrode.　The　detection　limit　for　B-GLU　after　2　h　of　incubation　was　0.07　U　L−1　when　4-methylumbelliferyl-β-D-glucuronide　was　used　as　the　fluorogenic　substrate　and　Br−　was　used　as　the　co-reactant.　B-GLU　recovery　rates　from　diluted　saliva　with　the　present　ECL　approach　were　adequate　(93–103　%)　and　similar　to　those　obtained　with　the　fluorescence　technique.　©　2025　Elsevier　B.V.