Objective: This study aimed at assessing the serial carboxyhemoglobin (COHb) levels in preterm infants during the first week of life and their variation with late-onset sepsis (LOS). Study Design: Infants with <37 gestational weeks were categorized into two groups according to the presence of culture proven LOS. Serial COHb levels were obtained during the first week of life, at the onset of the LOS episode, and upon blood culture negativity with response to antibiotics. Result: Overall 207 infants were enrolled. A LOS episode resulted in a significant increase in COHb levels (p < 0.001), which decreased to normal levels when the blood cultures were sterile (p < 0.001). At a cut of level of 1.35% COHb had a sensitivity of 56% and a specificity of 90% to confirm LOS (p < 0.001). Conclusion: In this study, we demonstrated an increase in COHb levels at the onset of LOS and a decrease with response to antibiotherapy.