Based on ompA gene sequence of Enterobacter sakazakii, the specific primer and probe were designed. An internal amplification control (IAC) was added and used to monitor the performance of reaction system. The assay could be used reliably to detect E. sakazakii genome DNA with the sensitivity of 1 pg. The detection limit for E. sakazakii was 1×10⁴ CFU when used the DNA extracted by water boiling as the template. With the use of ompA, the detection limit can reach 10³ copies. Through the standard curves of ompA, the quantification was linear between Ct and template copy number (R²=0.999). For the artificially contaminated powered milk with the concentration of 10 CFU/25 g, the E. sakazakii could be detected after 10 hours culture when used the DNA extracted by commercial kit and water boiling. The E. sakazakii fluorescence quantitative PCR assay provides reference data for optimization and modification of the standardized molecular biochemical approach of E. sakazakii.