Urine provides an alternative to blood plasma as a potential source of disease biomarkers. Exosomes was separated by ultracentrifuge at 200000 g in normal persons and non-small cell lung cancer (NSCLC) patients′ urine. For proteomic analysis of urinary exosome, 1D sodium dodecylsulfonate-polyacrylate gel electrophoresis(SDS-PAGE) was carries out and cut the gel 31 kDa-20 kDa bands in normal group and disease group′s. These gel blocks were subjected to in-gel trypsinization, and the extracted peptides were analyzed HPLC-CHIP-MS/MS. Approximately 24 unique proteins were identified in the UniProtKB/SWISS-PORT. The difference expression proteins were found in urinary exosome from NSCLC patients, including three fragment of the immunoglobulin kappa, two kinds of Ras related proteins, glutathione S-transferase A2, serum amyloid P-component precursor and phosphatidylethanolamine-binding protein 1.