Objective This study aimed to evaluate the cluster of differentiation (CD)64, CD16, CD11b, CD63 human leukocyte antigen-DR (HLA-DR), and CD62L leukocyte surface marker abnormalities using flow cytometry in the early diagnosis of late-onset neonatal sepsis. Methods Forty-four neonates were included in this study. Of them, 22 neonates with clinical late-onset neonatal sepsis were included in the study group, and the remaining 22 neonates without sepsis were considered the control group. Complete sepsis screening was performed. Additionally, monocyte and neutrophil surfaces marker were examined using flow cytometry. Results The expression of the leukocyte surface markers CD16 and CD64 on monocytes and neutrophils was significantly higher in the study group than in the control group (p < 0.05), while the CD63, CD62L, CD11b, and HLA-DR levels were similar to those in the control group (p > 0.05). Furthermore, receiver operating characteristic curve analysis indicated that neutrophil CD64 (nCD64) is a diagnostic marker for neonatal sepsis, with an area under the curve of 0.901. The CD64 and CD16, which are the respective leukocyte surface markers on neutrophils and monocytes, are useful tests in the early diagnosis of late-onset neonatal sepsis. Conclusion In addition to acute phase proteins, cell surface antigens such as CD16 and more specifically CD64 should be used in routine investigations for the early diagnosis of late-onset neonatal sepsis. Such use in combination with acute phase reactants can improve diagnostic accuracy.