Bladder cancer (BC) is the most common malignancy of the urinary tract. We developed a new and ELISA kit for detecting aberrantly glycosylated integrin α3β1 (AG31) in human urine. We analysed urine samples (n=408) of patients with BC, renal cell carcinoma (RCC), prostate cancer (PC), cystitis, nephritis, and prostatitis from two centres in China. The subjects in the validation groups (n=2317) were recruited from other centres in China between July 2012 and September 2013. Receiver operating characteristic (ROC) curves were used to determine diagnostic accuracy. AG31 levels in urine samples were significantly higher in patients with BC than in any of the control subjects. Moreover, elevated levels of AG31 in urine could distinguish BC from benign inflammatory diseases. Finally, the urinary AG31 test was much more sensitive and specific than the NMP22 test. Therefore, the urinary AG31 test will provide an ideal and assay for the detection of BCs.