Acute kidney injury (AKI) is a potential complication for children with congenital heart disease (CHD) after cardiopulmonary bypass (CPB) surgery. This study was designed to investigate and compare the predictive values of urinary biomarkers for AKI after CPB surgery in infants and young children and to determine the optimal timing of testing and the cutoff value for each biomarker. The study prospectively enrolled 58 CHD children 3 years of age or younger who were undergoing CPB surgery. Urinary neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), microalbumin (MA), N-acetyl--D-glucosaminidase (NAG), alpha 1-microglobulin (alpha 1-MG), and creatinine (UCr) were measured at baseline and at various time points after surgery. Children who experienced AKI had more complex cardiac surgical procedures as evaluated by Risk Adjustment for Congenital Heart Surgery 1 (RACHS-1), longer CPB and aortic clamping times, and worse clinical outcomes than those who did not. In the AKI group, all five urinary biomarkers increased substantially and peaked at 4 h after surgery. In contrast, in the non-AKI group, they increased slightly or had no significant changes during the first 24 h. All the biomarkers had the best predictive performances at 4 h after surgery. At this time point, NAG had the minimum area under the curve (AUC) (0.747), which was significantly lower than that of the others (AUC, 0.82-0.85; P < 0.05). The optimal cutoff value of each biomarker was 290 ng/mg UCr for NAGL, 1,477 pg/mg UCr for IL-18, 400 mg/g UCr for MA, 225 U/g UCr for NAG, and 290 mg/g UCr for alpha 1-MG. In conclusion, urinary NGAL, IL-18, MA, and alpha 1-MG had similar predictive performances for the early detection of AKI after CPB surgery in infants and young children.