The CD34+/CD38- immunophenotype is used to identify candidate hematopoietic stem cells (HSCs) and leukemia-initiating cells (LICs). However, the clinical significance of the CD34+/CD38-proportion in childhood acute lymphoblastic leukemia (ALL) is not well established. Here, we found that a high proportion of CD34+/CD38- cells from 112 patients with childhood ALL was negatively correlated with the outcome. Also the percentage of CD34+/CD38- cells was associated with clinical and biological features of patients with ALL. Further, a high proportion of CD34+/CD38- cells in childhood ALL was positively correlated with advanced risk subgroups, which could predict risk stratification by receiver operating characteristic (ROC) curve analysis. In addition, a larger tumor burden and lower survival rate were observed in mice injected with CD34+/CD38- cells, but not in mice injected with other fractions. Our data reveal that a high proportion of CD34+/CD38- cells is positively associated with a poor prognosis of childhood ALL, to further guide therapy of the disease.