Abstract Background The diagnosis of “suspected sepsis” in neonates in the NICU is one of the most common and challenging diagnoses due to the nonspecific signs of sepsis. therefore, laboratory investigation is necessary in cases of doubt or if there are risk factors. A promising biomarker: Presepsin (P-SEP), or Soluble Cluster of Differentiation 14 Subtype (sCD14-ST) is generated as a part of the body’s response to bacterial infection. It is effective in the early phase of inflammation, and it is detected as an early marker of sepsis in neonates. Aim and Objectives to evaluate the diagnostic value of presepsin in early neonatal sepsis and prognostic value for severity, mortality and hospital stay length. Subjects and Methods This prospective observational study include two groups, one group included 36 neonates with risk factors for early-onset sepsis, along with 20 uninfected neonates as the control group. Both groups were subjected to the calculation of Rodwell score, SNAP II score, serum presepsin levels, CRP measurement, and blood culture and assessed for clinical severity, mortality, and hospital stay length. Result Serum baseline presepsin was significantly elevated in sepsis group (P < 0.001), Presepsin showed the best diagnostic performance over Rodwell score (AUC 0.989; sensitivity of 93.8% and specificity of 95%). Presepsin levels on day 3 were significantly elevated in non-survived patients (P < 0.001), with better predictive performance for 28-day mortality and rapid progression to death than SNAP II score (AUC 0.880; sensitivity of 83.3% and specificity of 87.5%). Conclusion presepsin day 1 was useful diagnostic tools in culture proven early onset neonatal sepsis, presepsin at day 3 could be a good predictor for gram negative infections, 28-day mortality, and sepsis severity.