Objective This study aims to investigate the diagnosis and prognostic significance of miR-326 in childhood acute lymphoblastic leukemia. Methods 70 blood samples from childhood ALL patients and 30 blood samples from healthy volunteers were collected, and qRT-PCR was adopted to detect the relative expression level of serum miR-326. The ROC curve was used to analyze the diagnostic ability and prognostic value of miR-326 in childhood ALL. At the same time, Kaplan-Meier was also performed to draw survival curves to evaluate the survival of children with ALL patients with different expression levels of miR-326. Results Compared with the control group, the expression of miR-326 in the serum of childhood ALL patients was significantly down-regulated (P<0.05). The ROC curve showed an AUC value of 0.876 for the area under the curve, indicating that miR-326 has a good diagnostic value; overall survival analysis showed that low expression of miR-326 suggested a poor survival rate, and the ROC curve showed an AUC value of 0.909. Analysis of progression-free survival showed that low expression of miR-326 suggested a poor progression-free survival rate, and the ROC curve showed an AUC value of 0.815. This indicated that low expression of miR-326 is associated with a poor prognosis. Conclusion According to the expression of miR-326, it is obvious to distinguish children with ALL from healthy people. In addition, the low expression of miR-326 indicates a poor prognosis in children with ALL. Furthermore, miR-326 can be used as a diagnostic and prognostic marker for childhood ALL.