Objective To evaluate the significance of the CD64 index for the early diagnosis of neonatal sepsis. Methods The prospective case-control study was adopted to conduct the analysis. A total of 41 neonatal patients were divided into the documented sepsis group (n=14), clinical sepsis group (n=15), and control group (n=12). Venous blood specimens of all patients were collected with in 24 h after admission. The CD64 index was measured by the flow cytometry. Results Results of the flow cytometry showed that the CD64 indexes of the documented sepsis group, clinical sepsis group, and control group were 12.93±4.08, 9.51±2.21, and 2.98±0.72, respectively. Compared to the control group, the CD64 indexes of the documented sepsis group and clinical sepsis group were significantly higher (t=8.97, P=0.00; t=6.27, P=0.00). The CD64 index of the documented sepsis group was significantly higher than that of the clinical sepsis group (t=2.39，P=0.023). Conclusion Detection of CD64 contributes to the early diagnosis of neonatal sepsis, identifies the degree of infection, and provides an important evidence for appropriate clinical treatment.