Background: Neonatal sepsis is an important cause of neonatal deaths globally. Diagnosis of neonatal sepsis is established based on the clinical status of the newborn and microbiological tests of sepsis screen. Mid-phase markers of inflammation such as C-reactive protein (CRP) and serum procalcitonin (PCT) are considered useful and sensitive for early diagnosis. Most of the studies evaluating serum cord blood PCT as a diagnostic marker for neonatal septicemia have been carried out in peripheral venous blood with smaller sample sizes with the inclusion of neonates without considering perinatal sepsis scores (PSSs). Objective: To compare the diagnostic accuracy of cord blood PCT with venous blood PCT, alone and as part of sepsis screening parameters. Study Design: This hospital-based, prospective diagnostic accuracy study aimed to evaluate the diagnostic accuracy of serum PCT alone and in combination with other diagnostic modalities for the detection of early-onset neonatal sepsis in cord blood and to compare the diagnostic utility of PCT with neonatal sepsis screening parameters in venous blood and was conducted at a tertiary care center. Categorical/nominal variables were expressed as numbers and percentages and were analyzed using the chi-square test. The receiver–operating characteristic curve was drawn to determine the area under the curve (with a 95% confidence interval) for various parameters for the diagnosis of sepsis. Sensitivity, specificity, positive-predictive value (PPV), negative-predictive value (NPV), and diagnostic accuracy were calculated by using a 2 × 2 contingency table. All statistical analyses were done using SPSS trial version 20. Participants: A total of 247 newborns delivered with positive PSSs were enrolled. Intervention: In neonates with a positive PSS, cord blood serum PCT, sepsis screen, and blood culture were sent at the time of birth. They were observed for 72 hours for signs and symptoms suggestive of sepsis. Results: Statistical analysis of cord blood serum PCT for detecting blood culture-positive patients showed that PCT has a sensitivity of 44.4%, specificity of 86.4%, PPV of 33.3%, and NPV of 91.1%. Overall diagnostic accuracy is 80.9% indicating that cord blood PCT is a good test for identifying these patients ( p <.05). However, venous blood PCT failed to demonstrate similar results. Conclusion: Umbilical blood sampling protects neonates from the pain of venipunctures. Statistical comparison of cord blood PCT in relation to sepsis screen and/or blood culture and comparisons with the venous blood parameters in the same domains suggests better sensitivity and specificity than venous blood PCT. This early serological biomarker is valuable for early diagnosis and management while awaiting blood culture reports and helps in reducing the separation of neonates with suspected sepsis from their mother and thus helping in developmental supportive care.