OBJECTIVE Involvement of central nervous system in acute lymphoblastic leukemia (CNSL) remains one of the major causes of pediatric acute lymphoblastic leukemia (ALL) treatment failure. However, the current understanding of the pathological process of CNSL is still limited. This study aimed to better understand the protein expression in cerebrospinal fluid (CSF) of ALL and discover valuable prognostic biomarkers. METHODS CSF samples were obtained from ALL patients and healthy controls. Comparative proteomic profiling using label-free liquid chromatography-tandem mass spectrometry was performed to detect differentially expressed proteins. RESULTS In the present study, 51 differentially expressed proteins were found. Among them, two core clusters including ten proteins (TIMP1, LGALS3BP, A2M, FN1, AHSG, HRG, ITIH4, CF I, C2, and C4a) might be crucial for tumorigenesis and progression of ALL and can be potentially valuable indicators of CNSL. CONCLUSIONS These differentially expressed proteins of ALL children with central nervous system involvement and normal children may work as diagnostic and prognostic factors of ALL patients.