[Background] Cronobacter spp. are major foodborne pathogens that need to be intensively monitored for food safety. At present, with continuous development of the molecular detection technology, it is very important to develop simple and efficient detection methods for Cronobacter detection in food. [Objective] Develop duplex PCR detection kit for detecting Cronobacter and evaluate its detecting efficacy in food samples. [Methods] Duplex PCR reaction system was optimized and the components of the kit were confirmed. The main reaction reagents were made into powder pattern by freeze drying. Then the specificity, sensitivity, repeatability, shelf life and other performance indexes of the kit were evaluated. [Results] All Cronobacter standard strains and isolated strains were detected positive with two distinct bands in the target size, while all non-Cronobacter standard strains and isolated strains were detected negative without target band observed. The detection sensitivity of Cronobacter purified genomic DNA and pure cultures was respectively 2.3×10?1 ng/μL and 3.2×104 CFU/mL. Cronobacter in 65 food samples was detected. The detection result obtained by the kit was highly consistent with that obtained by conventional microbial detection method. The intra-batch and inter-batch testing repetition rates were all 100%. The kit had no efficacy loss after 120 h storage at 42 °C and 12 months at 4 °C. [Conclusion] The kit is stable, reliable, and applicable for rapid detection of Cronobacter in food.