The matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase associated lipocalin (NGAL) are shown to increase in an inflammatory situation. Based on our previous reports that NGAL can be detected in the urine of children with urinary tract infection (UTI), we also asked whether MMP-9/NGAL complex could be detected in the urine of children with UTI. This multicenter, prospective study was conducted between October 2009 and October 2010. Seventy-one patients with symptomatic culture proven UTI, 37 asymptomatic children with contaminated urine and 37 healthy children were recruited. Mean uMMP-9/NGAL/Cr levels were significantly higher in the UTI group than in the control group (p < 0.0001). According to ROC analysis, the optimal cut-off level was 0.08 ng/mg to predict UTI. Using a cut-off value, sensitivity and specificity were 98.6 and 97.3%, respectively. The mean levels of uMMP-9/NGAL/cr in the UTI group were also significantly higher than those in the contamination group (p < 0.0001). There was no statistically significant difference between contamination group and the control group (p = 0.21). The mean uMMP-9/NGAL/Cr in the UTI group were significantly higher before treatment than after treatment (p < 0.0001). The area under the curve was 0.997 (SE: 0.002, 95% CI: 0.993 to 1.001) for uMMP-9/NGAL/Cr. Urinary MMP-9/NGAL/Cr level was also correlated with positive urine nitrite test, positive urine leukocyte esterase reaction and renal scarring (p = 0.0001, p = 0.0001, p = 0.04, respectively) whereas was not correlated to leukocytosis and positive CRP level in serum. Urine MMP-9/NGAL/cr can be used as a diagnostic biomarker for UTI in children. Identification of NGAL-MMP-9/cr levels in the urine of suspected UTI patients may also be useful to differentiate between contamination and infection and for monitoring of treatment response in children.