Background The source of bacterial infection in neutropenic acute leukemia patients is detected in about 20–30% of cases. Bacterial cultures may require a long incubation period and risk false-positive and false- negative results. Therefore, biomarkers distinguishing septic febrile neutropenia from other etiologies in acute leukemia patients play the important role in patient assessment and treatment planning. This study aims to determine the role of procalcitonin (PCT) and presepsin (PSPN) in infectious complication in comparison to C-reactive protein (CRP) on the first and third day at the onset of febrile neutropenia in patients with acute leukemia. Methods Between June 2018 and February 2019, 60 acute leukemia patients with febrile neutropenia receiving chemotherapy. The 41 acute myeloid leukemia patients and 19 acute lymphoblastic leukemia patients were recruited in this study. Their ages ranged from 14 to 65 years. PCT and PSPN were measured and were compared to CRP at the onset of febrile neutropenia and after 48 hours. 20 patients had a fever of unknown origin (FUO) and 40 patients had a bacterial infection. Findings Our results showed that the values of these markers were higher in patients with infection than patients without. The area under the curve (AUC) of PCT were 0.931 and 0.813 on day one and three respectively, which was the best in determination of infection. The cut-off values of PCT were 1.27 and 1.23 ng/mL and the cut off values of PSPN were 1.75 and 2.9 μg/L in the successive days, their clinical sensitivities were high. PCT and PSPN were capable of distinguishing the cause of febrile neutropenia from the onset of infection and predicting its complications (p<0.05). The PSPN level couldn’t differentiate gram-positive or gram-negative bacterial infection. Significant differences were found between the mean values of the PSPN during the successive days in all patients and patients with bacteremia. This study illustrated a weak positive correlation between PCT and Sequential Organ Failure Assessment (SOFA) score, the negligible correlation between CRP and SOFA score and no significant correlation between PSPN and SOFA score. Interpretation PCT is an accurate biomarker in identifying infection in acute leukemia patients, its concentration is associated with the severity of bacterial sepsis. PSPN is superior to PCT for follow-up of patients.