Metabolomics shows tremendous potential for the early diagnosis and screening of cancer. For clinical application as an effective diagnostic tool, however, improved analytical methods for complex biological fluids are required. Here, we developed a reliable rapid urine analysis system based on surface-enhanced Raman spectroscopy (SERS) using 3D-stacked silver nanowires (AgNWs) on a glass fiber filter (GFF) sensor and applied it to the diagnosis of pancreatic cancer and prostate cancer. Urine samples were pretreated with centrifugation to remove large debris and with calcium ion addition to improve the binding of metabolites to AgNWs. The label-free urine-SERS detection using the AgNW-GFF SERS sensor showed different spectral patterns and distinguishable specific peaks in three groups: normal control (n = 30), pancreatic cancer (n = 22), and prostate cancer (n = 22). Multivariate analyses of SERS spectra using unsupervised principal component analysis and supervised orthogonal partial least-squares discriminant analysis showed excellent discrimination between the pancreatic cancer group and the prostate cancer group as well as between the normal control group and the combined cancer groups. The results demonstrate the great potential of the urine-SERS analysis system using the AgNW-GFF SERS sensor for the noninvasive diagnosis and screening of cancers.