Abstract: Objective A detection assay for alive Shigella in food was developed by using of ethidium monoazide bromide (EMA) real-time fluorescence PCR technology, which is convenient, rapid, specific and sensitive. Methods The conserved ipaH gene was chose as the specific primer and probe to detect Shigella. Samples were pretreated with EMA at different concentrations and irradiating times. The detection sensitivity, specificity and stability were evaluated by using 31 Shigella strains, 26 Listeria monocytogenes strains, 24 Salmonella strains, 25 Vibrio parahemolyticus strains, 11 Cronobacter sakazakii strains, 10 Enteropathogenic Escherichia coli strains, and 1 E. coli strain, and the comparisons of the specificity and stability in detection of 30 simulated samples were made between newly developed EMA real-time fluorescence PCR assay and routine culture. Results The Ct value of EMA real-time fluorescence PCR for alive Shigella was 32.10-3.19log (bacteria load).The lowest limit of detection was 2.20 CFU per reaction. More than 99.97% of PCR reaction for dead strains was inhibited. The Ct values of 31 Shigella strains were between 15.04 and 26.54, while 97 non-Shigella strains had Ct values35 or had no Ct values (one horizontal line appeared). The variation coefficient of Ct was less than 3 when same samples were tested every other day for five times. The results of the detections of 30 simulated samples were consistent by either using newly developed EMA real-time fluorescence PCR or routine culture, but the test time was shortened from 3-5 days (routine culture)to less than 7.5 h. Conclusion The newly developed EMA real-time fluorescence PCR for alive Shigella is rapid, convenient, specific and sensitive, it is recommended to use it in food inspection.