Hydrated　electrons　were　generated　at　boron　doped　diamond　electrodes　in　fully　aqueous　solutions　during　cathodic　pulse　polarization　and　utilized　for　the　electrochemiluminescence　(ECL)　determination　of　alkaline　phosphatase　(ALP)　by　adapting　one　of　the　most　common　fluorescent　methods.　In　this　approach,　highly　fluorescent　4-methylumbelliferyl　(MU)　is　enzymatically　generated　from　non-fluorescent　4-methylumbelliferyl　phosphate　(MUP)　substrate　and　chemically　excited　in　the　presence　of　oxidizing　radicals　and　reducing　hydrated　electrons.　A　novel　time-resolved　internal　standard　strategy　is　presented　to　counter　matrix　effects　to　certain　degree　and　to　lower　signal　variation　among　concurrent　measurements.　In　this　unique　approach,　the　cathodic　ECL　signals　of　MU　and　Tb(III)-chelate　were　separated　on　the　basis　of　their　exceptionally　different　excited　state　lifetimes.　This　enabled　more　precise　determination　of　ALP　by　simply　measuring　the　ratio　of　ECL　emissions　during　and　after　cathodic　excitation　pulse　with　simple　two-electrode,　single　photomultiplier　tube　setup.　The　ratiometric　ECL　signals　in　the　absence　and　presence　of　1.0　%　serum　(v/v)　were　interchangeable　whereas　ratiometric　signal　recovery　in　the　presence　of　10.0　%　serum　was　∼85　%.　©　2024