BACKGROUND: Acute lymphoblastic leukemia (ALL) is a prevalent hematologic malignancy that primarily affects children. The diagnosis and treatment of pediatric ALL remain challenging. This study aimed to identify differential lipids and metabolites that may hold potential for improving ALL treatment. METHODS: In this retrospective case-control study, serum samples obtained from children with ALL and healthy controls were analyzed. Serum lipidome and metabolome alterations of ALL were analyzed by comparing pediatric patients with ALL with healthy controls based on liquid chromatography high-resolution mass spectrometry analysis of serum lipidomic and metabolomic signatures. RESULTS: We identified 2,298 lipid features in the serum. Among them, 72 (3.13%) differed significantly in pediatric patients with ALL compared to healthy controls. Notably, sphingolipids (ceramide and sphingomyelin) and phospholipids exhibited the most pronounced changes. Targeted analysis of ceramides revealed significantly elevated levels of Cer 18:0 and Cer 20:0 in the serum of pediatric patients with ALL. Additionally, gut microbial-related lipids (such as sulfonolipids and fatty acid esters of hydroxy fatty acids) showed significant alterations. Metabolomic analysis identified 15 differential metabolites, indicating disrupted nucleotide and amino acid metabolism. Furthermore, the dysregulated lipids and metabolites correlated with various blood indicators, with ceramide and nucleosides positively associated with white blood cell count but negatively correlated with hemoglobin and platelet. CONCLUSION: These findings shed light on abnormal molecular signatures contributing to pediatric ALL and may serve as potential biomarker panel for therapy of ALL.