Aim: High-dose methotrexate (HD-MTX) therapy is commonly used in acute lymphoblastic leukemia (ALL) which need drug monitoring. This study was to analyze the influence factors and prognostic role of the plasma MTX concentration in ALL. Methods: 1435 HD-MTX courses of 246 childhood ALL were enrolled. MTX doses were 3 g/m2 for low-risk (LR) group and 5 g/m2 for intermediate or high-risk groups. The target 24-hours (24h) MTX concentrations were set at 33 μmol/L for LR and 65 μmol/L for non LR group. Results: The median 24h MTX concentrations were 42.0 μmol/L for LR and 70.0 μmol/L for non LR group. Only SLCO1B1 genotype in the LR group and age in the non LR group was associated with the 24h MTX concentrations. The survival results were comparable between patients with or without courses failed to reach the target 24h MTX concentration. MTX excretion delay was observed in 211/1435 (14.7%) courses of 125 patients, which more commonly caused MTX-induced toxicities. All the 6 CNS relapses occurred in patients with MTX excretion delay, while in those without did not have any type of CNS relapse (6/119 vs. 0/121, P=0.015). Patients with more than 20% of MTX excretion delay courses had significant worse survival (83.8%±5.0% vs. 93.7%±1.8% for EFS, P=0.014, and 88.8%±4.3% vs. 97.2%±1.1% for OS, P=0.010). Conclusions: Achieving the target 24h MTX concentration during HD-MTX therapy is not the useful indicator to predict the survival of ALL patients. MTX excretion delay is an independent factor for predicting the CNS relapses and worse prognosis.