Exosomes are membrane-enclosed phospholipid extracellular vesicles which can act as mediators of intercellular communication. Although the original features endow tumor-derived exosomes great potential as biomarkers, efficient isolation and detection methods remains challenging. Here, we presented a two-stage microfluidic platform (ExoPCD-chip) which integrates on-chip isolation and in situ electrochemical analysis of exosomes from serum. To promote exosomes capture efficiency, an improved staggered Y-shaped micropillars mixing pattern was designed to create anisotropic flow without any surface modification. By combining magnetic enrichment based on specific phosphatidylserine-Tim4 protein recognition with a new signal transduction strategy in a chip for the first time, the platform enables highly sensitive detection for CD63 positive exosomes as low as 4.39×103 particles/mL with a linear range spanning 5 orders of magnitude, which is substantially better than the existing methods. The reduced volume of samples (30 μL) and simpler affinity method also make it ideal for rapid downstream analysis of complex biofluids within 3.5 h. As a proof-of-concept, we performed exosomes analysis in human serum and liver cancer patients can be well discriminated from the healthy controls by the ExoPCD-chip. These results demonstrate that this microfluidic chip may serve as a comprehensive exosome analysis tool and potential non-invasive diagnostic platform.