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The present study establish a simple method for determining the concentration of 6β-hydroxycortisol and cortisol in urinary at same time, to determined the urinary 6β-hydroxycortisol/cortisol ratio, a marker for CYP3A activity in 13 Chinese children acute lymphoblastic leukemia patients compare with 114 health children. First-morning urine specimen was collected from each subject for the quantification of urinary concentrations of cortisol and 6β-hydroxycortisol. In this study, the association of the 6β-OHC/cortisol ratio was higher in children acute lymphoblastic leukemia patients than in health children, while the 6β-hydroxycortisol/cortisol ratio is no significant different between subgroups in health children (P). These findings suggest that high CYP3A4 activity may be a risk factor for children acute lymphoblastic leukemia risk. 6β-hydroxycortisol/cortisol ratio could be consided as a probe childhood acute lymphoblastic leukemia.