Objective To explore the clinical significance of neutrophil CD64 in early diagnosis and evaluation of disease condition of neonatal septicemia. Methods This case-control study included 18 neonates with suspected septicemia (septicemia group) and 32 neonates without infection (non-infection group) in our hospital from June 2015 to November 2015. The umbilical venous blood at birth and peripheral venous blood at 0-12 h were collected from all the cases for blood culture and detection of CD64 level. The septicemia group was given recheck of CD64 at 48-72 h, and the non-infection group was given recheck of CD64 at 72 h, and the levels of CD64 were compared. Results The CD64 levels of both umbilical venous blood and peripheral blood in the septicemia group were significantly higher than those in the non-infection group (t = -3.15 and -3.35, P = 0.010 and 0.004). However, the CD64 level of umbilical venous blood had no significant change from that of peripheral blood at 0-12 h in the septicemia group (t = -0.507, P = 0.623). Conclusions Neutrophil CD64 has certain clinical application value in early diagnosis of neonatal septicemia. Neutrophil CD64 of umbilical cord blood can be used as a credible marker for the diagnosis of intrauterine bacterial infection and early onset of septicemia.