Objective To investigate the occurrence of healthcare-associated infection (HAI) complicated with sepsis in hospitalized children with acute leukemia during chemotherapy, and explore its pathogenic characteristics and risk factors. Methods From January 2021 to June 2022, 819 children who were diagnosed with acute leukemia in the pediatric department of a tertiary first-class hospital were selected as the study subjects. The nested case-control study method was adopted. Children with sepsis during the monitoring period were in the case group, and children with the same type of leukemia during the same hospitalization period were randomly selected as the control group based on 1∶2 ratio. Differences in basic information and research indicators between two groups of children were compared. Risk factors for sepsis related to leukemia chemotherapy, distribution and antimicrobial susceptibility of pathogens from blood specimens were analyzed. Results Among 819 children with acute leukemia, 51 had sepsis, with an incidence of 6.23%. Paired in a 1∶2 ratio, 102 children were included in the control group. 29 strains of pathogens were isolated from blood specimens, including 21 strains (72.41%) of Gram-negative bacteria (mainly Escherichia coli) and 8 strains (27.59%) of Gram-positive bacteria (mainly Streptococcus midis). Use of glucocorticoids (OR=13.20, 95%CI: 3.42-155.81), absolute neutrophil count < 0.1×10⁹/L (OR=38.09, 95%CI: 12.06-357.61) and serum albumin < 35 g/L (OR=7.61, 95%CI: 2.11-39.76) were independent risk factors for sepsis in children with acute leukemia during chemotherapy (all P < 0.05). Conclusion It is necessary to strengthen the monitoring, prevention and control, and follow-up of the infection in children with acute leukemia during chemotherapy, especially for children who use glucocorticoids, with low absolute neutrophil count and low serum albumin level, so as to reduce the occurrence of HAI-associated-sepsis.