The distinction between viable and dead cells was a major issue in detection of pathogenic microbe in foods especially when foods had been subjected to thermal processing. The performance of a loop-mediated isothermal amplification (LAMP) assay with aid of ethidium monoazide (EMA) for detecting viable Escherichia coli O157 in raw milk was presented in this paper. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. LAMP can only amplified DNA of viable Escherichia coli O157 because EMA selectively penetrated dead cells and covalently bound to DNA, detection limit level for artificially contaminated raw milk samples by the EMA-LAMP assay was 440 cfu/mL corresponding to 3–5 cells per reaction tube, while the detection level by EMA-PCR was 4.4×104 cfu/mL. In conclusion, EMA-LAMP had offered a novel assay for distinction between viable and dead cells with promising application in food safety detection.