Neonatal sepsis is a life-threatening disorder among infants, which is associated with high morbidity and mortality. In recent years, although the management of newborns has improved, early diagnosis and treatment of neonatal earlyonset sepsis (NEOS) are the major challenges for neonatologists in Neonatal intensive care units (NICUs). We assessed the diagnostic value of interleukins-35 (IL-35) in diagnosis of NEOS. A prospective process evaluation study was carried out in the NICU of Akbar-Abadi and Ali-Asghar Hospitals affiliated to Iran University of Medical Sciences from 2017 to 2018. Eighty hospitalized neonates with clinical suspicion of sepsis in the first 72 h of life entered the study by convenience or accidental sampling. Routine hematology tests regarding sepsis diagnosis were performed after admission. All subjects were assigned into two groups: newborns with proven sepsis and newborns with suspected sepsis. IL-35 levels in septic and unlikely infected hospitalized newborns were measured using Elisa Kit (ZellBio GmbH. Cat. No; ZB-10042C-H9648, Human Interleukin 35), and the results were compared. The mean IL-35 in serum samples from septic neonates was significantly higher than this variable in samples from unlikely infected newborns (13.41 ± 2.4 vs. 9.02 ± 2.6 pg/ml; p < 0.0001). The area under receiver-operating characteristic (ROC) curve for IL-35 was 0.895 (CI 95% = 0.826-0.963; p = 0.0001). The result of study showed serum IL-35 had a moderate accuracy (AUC = 0.895) for the diagnosis of NS. Therefore, serum IL-35 could be suggested as a predictive biomarker in neonatal early-onset sepsis. Further studies with larger sample size are strongly suggested.