To search for biomarkers critical for bladder carcinoma diagnosis and prognosis, secreted proteomes of highly malignant U1 and pre-malignant U4 cell lines were initially analyzed. Proteins in the culture media of the U1 and U4 cell lines were systematically examined by SDS-PAGE combined with MALDI-TOF MS. Among them, expression of pro-u-plasminogen activator (pro-u-PA) was confirmed by Western blot analysis and further evaluated. In analyzing urine samples from bladder cancer patients and normal subjects, we established a statistically significant relationship between the low level and absence of pro-u-PA in urine with high stages and grades of the tumor samples. Constitutive expression of Ras dominant negative protein led to increased expression of pro-u-PA in culture media, indicating that the loss of pro-u-PA is associated with oncogenic transformation. Analysis of cancer-secreted proteomes can be a feasible, non-invasive and efficient strategy for searching potential bladder tumor biomarkers. Our work also has identified the loss of pro-u-PA in urine as potential marker of more advanced bladder carcinoma.