Objective To prospectively explore the value of urinary neutrophil gelatinase-associated lipocalin(uNGAL) determined at different time points in early diagnosis of cardiac surgery-associated acute kidney injury(AKI). Methods A total of 111 hospitalized patients undergone cardiac surgery were enrolled into this study from October 2011 to May 2012. The urinary samples were collected before cardiac surgery and at 0, 6, 12, 24, 48, 72 h after surgery, and uNGAL was immediately measured by ARCHITECT i2000. According to AKI criteria of Kidney Disease Improving Global Outcomes(KDIGO) clinical practice guidelines on AKI, the subjects were divided into the AKI group and non-AKI group. Results Twenty-four of the 111 patients developed acute kidney injury(21.6%). Compared with non-AKI group, the uNGAL level of AKI group was dramatically increased at 0 h after surgery〔54.20(9.60-452.10) vs 8.95(3.03-24.65), P<0.001〕, and was also significantly higher at 6 h after surgery〔35.50(10.60-80.10) vs 12.20(6.30-38.40), P<0.05〕. The 0h after surgery-to-baseline ratio of uNGAL was significantly higher in AKI group than in non-AKI group(2.72 vs 1.09, P<0.01). The area under the receiver operating characteristic(ROC) curve, the cutoff value, the sensitivity and specificity for 0 h uNGAL after surgery were 0.852(0.777-0.926, P<0.001), 28.25 ng/mL, 91.7% and 77.4%, for 6 h uNGAL after surgery were 0.650(0.500-0.651, P<0.026), 26.50 ng/mL, 50.0% and 65.1%, for 0 h uNGAL after surgery-to-baseline ratio were 0.752(0.642-0.862, P=0.001), 1.95, 93.3% and 64.5%, respectively. Conclusion The uNGAL can be used as an early biomarker for prediction of cardiac surgery-associated acute kidney injury(AKI). The uNGAL 0 h after surgery was the best point for diagnosis of AKI.