Tumor-derived exosomes, which contain RNA, DNA, and proteins, are a potentially rich non-invasive source of biomarkers. However, no efficient isolation or detection methods are yet available. Here, we developed a microfluidic Raman biochip designed to isolate and analyze exosomes in situ. Anti-CD63 magnetic nanoparticles were used to enrich exosomes through mixing channels of a staggered triangular pillar array. EpCAM-functionalized Raman-active polymeric nanomaterials (Raman beads) allow rapid analysis of exosome samples within 1 h, with a quantitative signal at 2230 cm-1. The limit of detection of this biochip approaches 1.6 × 102 particles per mL with 20 μL samples. The newly developed biochip assay was successfully applied in the determination of exosomes from clinical serum samples. Thus, this novel device may have potential as a clinical exosome analysis tool for prostate cancer.