Neonatal sepsis (NS) continues to be one of the most significant causes of neonatal morbidity and mortality. Early identification of Neonatal sepsis is a major diagnostic problem because of the nonspecific clinical signs and limitations of the current diagnostic procedures. Neutrophil CD64 expression has been proposed as a diagnostic test for evaluation of infection and sepsis. We compared the diagnostic utility of neutrophil CD64 expression with IL-6, IL-8, TNFalpha and CRP assays. Peripheral blood samples were taken from 25 neonates classified into two groups; proven NS (n = 15), clinical NS (n = 10) and healthy newborns (n = 10). CD64 expression was analysed by flowcytometry, while serum level of interleukins (IL-6, IL-8), and TNFalpha was determined by ELISA. Expression of CD64 was significantly enhanced in the groups with proven sepsis and clinical NS as compared to the controls (P < 0.05). Similary, TNFalpha, IL-6, IL-8 and CRP levels were significantly elevated in the groups with sepsis and clinical NS as compared to the controls (P < 0.05). Our data indicate that, in addition to serum levels of interleukins (IL-6, IL-8), and TNFalpha, expression of CD64 on neutrophils by flowcytometry could be useful as an indicator of NS due to its early appearance, sensitivity and specificity (96%). In conclusion, neutrophil expression of CD64 is a useful diagnostic tool for early detection of neonatal sepsis. The assay is rapid, easy and reliable.