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ObjectiveTo establish a real-time quantitative polymerase chain reaction （PCR） method for rapid and accurate identification of Cronobacter spp. in food samples and artificially contaminated samples.MethodsPrimers and probes were designed based on the conserved region of gyrB of Cronobacter spp. DNA. The method was verified using a specificity test， absolute sensitivity test， relative sensitivity test， and anti-interference test. Detection sensitivity was determined using artificially contaminated samples.ResultsThe method established in this study could specifically amplify seven kinds of Cronobacter spp.， but not the other Enterobacter species closely related to it and other pathogens common in food， suggesting that this method has good anti-interference ability. The absolute sensitivity was 1-10 pg and the relative sensitivity was 103 CFU/mL using Cronobacter sakazakii. It had good anti-interference ability at the genome and culture level. The sensitivity of artificially contaminated samples could reach 100 CFU/mL after incubation at 36 ℃ for 24 h.ConclusionThe real-time PCR method developed in this study is rapid， specific， sensitive， and stable for the detection of Cronobacter spp. in infant formula food samples and can provide technical reference for the detection of Cronobacter spp. in infant formula food.