Glycolysis,　a　pivotal　step　in　yeast　metabolism,　plays　an　indispensable　role　as　a　carbohydrate　utilization　process　crucial　for　cellular　survival.　Developing　advanced　technologies　to　elucidate　this　fundamental　physiological　process　holds　significant　scientific　implications.　Electrochemiluminescence　(ECL)　imaging　exhibits　the　advantage　of　negligible　background　interference　and　facilitates　straightforward　visualization,　thereby　conferring　significant　value　in　biomolecular　observation.　In　this　study,　we　present　an　ECL　imaging　method　for　investigating　yeast　metabolism　by　utilizing　the　endogenetic　NADH　as　an　efficient　coreactant　for　ECL　generation.　The　yeast　glycolysis　process　drives　the　conversion　of　NAD+　to　NADH,　resulting　in　enhanced　ECL　response　as　well　as　the　increased　brightness　of　ECL　images　that　can　be　used　for　quantification　of　yeast　activity.　There　was　a　linear　correlation　between　the　reciprocal　of　both　the　gray　value　of　ECL　image　and　yeast　concentration　within　the　range　of　6.25　×　106　-　6.25　×　108　CFU/mL.　Due　to　the　highly　efficient　coreactant　behavior　of　NADH,　our　method　demonstrated　excellent　selectivity　with　minimal　interference.　Furthermore,　we　employed　this　approach　to　investigate　some　toxic　inhibitors　on　yeast　metabolism,　yielding　reliable　results.　This　ECL　imaging　method　not　only　avoids　the　use　of　additional　coreactants　but　also　provides　a　sensitive　and　intuitive　approach　for　monitoring　yeast　metabolism,　demonstrating　great　potential　in　revealing　various　complex　biological　processes.　©　2024　American　Chemical　Society.