Our aim was to evaluate biochemical markers in plasma (NGAL, CysC) and urine (NGAL, KIM-1) in children’s early onset of acute kidney injury after congenital heart defect surgery using cardiopulmonary bypass. This study prospectively included 100 children with congenital heart defects who developed AKI. Patients with acute kidney injury had significantly higher CysC levels 6 and 12 h after cardiac surgery and plasma NGAL levels 2 and 6 h after cardiac surgery. The best predictive properties for the development of acute kidney injury are the combination (+CysCpl or +NGALu) after 12 h and a combination (+CysCpl and +NGALu) 6 and 24 h after cardiac surgery. We showed that plasma CysC and urinary NGAL could reliably predict the development of acute kidney injury. Measurement of early biochemical markers in plasma and urine, individually and combination, may predict the development of cardiac surgery-associated acute kidney injury in children.