Background:  This study designed to evaluate the level of IL-27 in umbilical cord blood and the possibility of its use as a predictor for diagnosis of Early Onset Neonatal Sepsis (EONS). Methods: This observational analytic study was conducted by enrolling newborn infants born to pregnant women with antenatal risk factors for sepsis. The infection group that included both suspected and confirmed EONS occurred ≤72 hours after delivery compared to non-infection group. Blood samples were collected from the umbilical artery after cord clamping for biomarkers detection and after 24 hours from venous blood. The concentrations of IL-27 and procalcitonin (PCT) were measured by sandwich ELISA assay. Results: A total of 124 neonates were enrolled, 48 (38.7%) neonates were identified in infection group either suspected or confirmed EONS. A significantly higher levels of IL-27 was found in cord blood of babies in the infection group (p<0.01).  At 24h after birth, the IL-27 continued to show significantly higher levels in infection group (p<0.01) compared to non-infection group. The IL-27 showed an increased risk of EONS with an odds ratio of 9.13 and p<0.01. The IL-27 through ROC curve analysis showed a better performance in distinguishing neonates with true infection from neonates without infection. A combined performance of IL-27 with PCT in the cord blood and at 24 hours of life showed greater prediction of EONS with p <0.01 for each. Conclusion: The IL-27 level in the blood of the umbilical cord may offer a significant predictive tool for EONS either alone or more significantly in combination with PCT.