Enterobacter sakazakii was an emerging food-borne pathogen associated with meningitis, sepsis and necrotizing enterocolitis, especially in neonates with high potential danger. The conventional detection methods were time-consuming and operated arduously, sometimes gave ambiguous signals. Increasing of reports showed the infant formula was the main infection vehicle. Consequently, it was important that improvement of methods for earlier detection and confirmation of presumptive E.sakazakii in infant formula to control and guard against the epidemic diseases due to E.sakazakii. In this study, PCR assay was developed based on ompA and α-1, 4-glusidase genes. Expected fragments(469 bp and 673 bp)were produced from 8 strains of E. sakazakii including E.sakazakii ATCC51329 after PCR amplification, but not from 70 strains of other bacteria. The sensitivity is 10¹ cfu/mL and 10² cfu/mL by signal-PCR using ESSF-ESSR and ESF-ESR as primers respectively in pure culture, sensitivity of dual-PCR is 10³ cfu/mL. Detection limit in artificially contaminated infant formula is 10² cfu/mL and 10³ cfu/mL by single-PCR with ESF-ESR and ESSF-ESSR respectively. To investigate whether the presence of other bacteria in infant formula have any effect on the sensitivity of PCR, two sets of experiment were designed. Different levels of other bacterial do not affect the PCR detection limit, which indicates the primers are species-specific for E.sakazakii. The investigation of actual samples shows that PCR assay is consistent with FDA standard method. The new method developed in the study can be used widely to detect the presence of E.sakazakii in infant formula with higher sensitivity and specificity than conventional methods.