This study was aimed to explore the clinical diagnostic significance of cerebrospinal fluid detection with flow cytometry (FCM) in children acute lymphoblastic leukemia (ALL) accompanied by central nervous system leukemia (CNSL). The 75 cerebrospinal fluid (CSF) samples of children with de novo ALL were detected by FCM, conventional cytology (CC), CSF routine tests and cytochemical examination. The results showed that among 75 de novo ALL, median age of onset was 5 (1 - 14) years old, ratio of male/female was 1.3:1. According to CCLG2008-ALL risk group protocol, there were 38 cases of standard risk (50.7%), 22 cases of intermedia risk (29.3%), 15 cases of high risk (20%). The results of CSF detection showed FCM(+)CC(+) 5 cases (6.7%), FCM(+)CC(-) 8 cases (10.7%) and FCM(-)CC(-) 62 cases (82.7%). According to CNS status classified, there were 3 cases of CNS-2, 2 cases of CNS-3 in FCM(+)CC(+) group there were CNS-1 7 cases, CNS-2 1 case in FCM(+)CC(-) groups, there were CNS-1 60 cases, CNS-2 1 case, CNS-3 1 case in FMC(-)CC(-) group. There was no significant difference in the distribution of the CNS-1, -2, -3 among the FCM(+)CC(+), FCM(+)CC(-), FCM(-)CC(-) groups (P > 0.05). It may be related to the fewer samples. There was higher consistency between results of FCM and CC on CNS-2, -3 diagnoses. FCM increased CC diagnosis positive rate to 20%. Meanwhile, there was no malignant cell in the smear, and no abnormal lymphocytic immunophenotype could be seen in CSF samples from 9 cases of viral encephalitis. It is concluded that cerebrospinal fluid detection with FCM has higher sensitivity, which is an important supplement to CSF routine detections and has significant useful value in diagnosis of children CNSL.