Cell-free microRNAs (miRNAs) stably and abundantly exist in body fluids and emerging evidence suggests cell-free miRNAs as a novel class of noninvasive disease biomarkers. In this study, we hypothesized that the quantitative detection of the oncogenic miR-106b-25 cluster in urine could be a useful clinical biomarker for bladder cancer (BCa). Three members of the miR-106b-25 cluster (miR-106b, miR-93 and miR-25) were quantified by real-time RT-PCR in urine supernatant of 112 BCa patients and 78 age-matched controls. In our study, the urinary levels of miR-106b were significantly higher in BCa patients than controls (P<0.001). No significant difference was observed in the urinary levels of miR-93 and miR-25 between two groups. Furthermore, the levels of urinary miR-106b were significantly reduced in postoperative samples compared with the levels in the preoperative samples (P=0.007). With respect of clinicopathological characteristics, the level of urinary miR-106b was associated with advanced tumor stage. Receiver operating characteristic (ROC) analysis revealed that urinary miR-106b had considerable diagnostic accuracy, yielding an AUC (the areas under the ROC curve) of 0.802 with 76.8% sensitivity and 72.4% specificity in differentiating BCa from controls. In conclusion, our data indicate that urinary cell-free miR-106b might provide new complementary tumor biomarkers for BCa.