Background The most common malignancy in children is acute lymphoblastic leukemia (ALL). This study aimed to explore KLK10 mRNA expression as a potential diagnostic biomarker for ALL in children and to examine the effect of chemotherapy on KLK10 mRNA expression following the induction and after three months of receiving chemotherapy. Methods In this prospective study, total RNA was extracted from blood samples of 23 pediatric ALL patients on diagnosis, after one month and three months of receiving chemotherapy. Healthy pediatric volunteers (n = 12) were selected as control individuals. After cDNA synthesis, KLK10 mRNA gene expression levels were quantified using quantitative real-time PCR (qRT-PCR). Results KLK10 mRNA expression levels were significantly decreased in leukemic cells compared to their levels in cells of normal blood samples (p = 0.0001). KLK10 expression levels in ALL patients after one month and three months of receiving chemotherapy decreased compared to normal blood samples (p < 0.0001 and p = 0.0175 respectively). The expression level of KLK10 mRNA in ALL patients after one month of chemotherapy was decreased compared to their level on diagnosis (p = 0.4413). KLK10 mRNA expression levels in ALL patients after three months of chemotherapy were increased compared to their level on diagnosis (p = 0.0602). The ROC curve illustrated that KLK10 mRNA expression could very efficiently discriminate ALL patients from normal counterparts (AUC=0.886, 95% CI [0.7720–1.000], SE = 0.0582, p = 0.0004). Conclusion KLK10 mRNA expression could serve as a potential diagnostic molecular biomarker for ALL in children.