Exosomes are lipid bilayer extracellular vesicles secreted by various types of cells and inherit abundant molecular information from parental cells. Tumor-derived exosomes have been widely recognized as noninvasive biomarkers for early cancer diagnosis and surveillance, but the separation of intact exosomes and detection of exosomal proteins remain challenging. Herein, we proposed a microfluidic chip for specific exosome isolation, integrated with sensitive quantification by a novel PTCDI-aptamer signal switch strategy. To enhance the capture efficiency, an alternating drop-shaped micropillar array was designed to assist the capture of tumor-derived exosomes by Tim4-modified magnetic beads (Tim4 beads) on the chip. Following capture, a chelating agent can easily elute intact exosomes which were further used for profiling exosomal surface proteins by the multiplexed fluorescence turn-on approach. Profiting from the efficient on-chip enrichment of the Tim4 beads and superior fluorescence signal transduction strategy, the detection limit of the analysis platform for HepG2 exosomes is as low as 8.69 × 103 particles/mL with a wide linear range spanning 6 orders of magnitude. Meanwhile, the proposed platform could recognize subtle changes in protein levels on the exosomal surface from various cell lines. More importantly, this strategy is successfully applied to analyze exosomes in human serum to distinguish liver cancer patients from healthy individuals. Combined analysis of different types of biomarkers on the exosomal membrane surface can greatly improve the accuracy of cancer type identification and disease monitoring. We hope that this convenient, rapid, and sensitive platform may become a powerful tool in the field of exosome analysis and early cancer screening.