Foodborne or water-borne pathogens pose great threats to human beings and animals. There is an urgent need to detect pathogens with cheap, rapid and sensitive point-of-care diagnostic assays. Herein, we report the electrochemiluminescent (ECL) behaviors of NaBiF₄ : Yb³⁺/Er³⁺ upconversion nanoparticles (UCNPs) which were synthesized via a fast and environment-friendly method at room temperature for the first time. The UCNPs together with K₂S₂O₈ exhibit high ECL intensity and stable cathodic signals. Further, the Au nanoparticles (Au NPs) and Anti-E. coli O157 : H7 antibody were assembled on the surface of UCNPs successively to construct a novel ECL immunosensor for the detection of deadly E. coli O157 : H7. The as-prepared ECL immunosensor reveals high sensitivity to E. coli O157 : H7 in a linear range of 200–100 000 CFU mL⁻¹, and the minimum detection limit could reach up to 138 CFU mL⁻¹. The designed UCNP-based biosensor demonstrates high specificity, good stability and remarkable repeatability, and the strategy will provide a sensitive and selective method for rapid detection of E. coli O157 : H7 in food safety and preclinical diagnosis.