Background:　Escherichia　coli　(E.　coli)　is　a　model　microorganism　extensively　used　in　microbial　research　due　to　its　well-characterized　physiology　and　genetic　tractability.　Monitoring　its　physiological　indicators,　such　as　metabolic　activity　and　stress　responses,　is　essential　for　understanding　microbial　life　processes.　However,　existing　methods　often　lack　sensitivity,　rapidity,　and　biocompatibility.　This　study　addresses　these　limitations　by　combining　electrochemiluminescence　(ECL)　biosensing　with　a　biocompatible　interface,　to　enable　precise　and　efficient　monitoring　of　E.　coli＇s　physiological　state.　Results:　This　study　presents　a　novel　ECL　biosensor　that　leverages　the　biocompatibility　of　bovine　serum　albumin　(BSA)　with　E.　coli.　The　biosensor　employs　horseradish　peroxidase　(HRP)　and　4-chloro-1-naphthol　(4-CN)　to　induce　a　precipitation　reaction　that　quenches　the　ECL　signal　of　CdS–K2S2O4,　enabling　the　sensitive　detection　of　endogenous　hydrogen　peroxide　(H2O2)　produced　by　E.　coli.　The　sensor　exhibits　excellent　linearity,　stability,　and　reproducibility,　with　a　detection　limit　of　54　nM　for　H2O2.　Additionally,　the　biosensor　effectively　distinguishes　between　antibiotic-resistant　and　sensitive　strains　of　E.　coli,　demonstrating　its　potential　for　comprehensive　physiological　assessment.　Significance:　This　innovative　ECL　biosensor　represents　a　significant　advancement　in　microbial　monitoring,　combining　high　sensitivity,　rapidity,　and　biocompatibility,　offering　a　powerful　tool　for　comprehensive　physiological　monitoring　in　bioanalytical　sciences.　Its　potential　applications　in　biotechnology,　food　industry,　and　drug　research　highlight　its　practical　relevance.　©　2025