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