Monitoring targeted therapy in real time for cancer patients could provide vital information about the development of drug resistance and improve therapeutic outcomes. Extracellular vesicles (EVs) have recently emerged as a promising cancer biomarker, and EV phenotyping shows high potential for monitoring treatment responses. Here, we demonstrate the feasibility of monitoring patient treatment responses based on the plasma EV phenotypic evolution using a multiplex EV phenotype analyzer chip (EPAC). EPAC incorporates the nanomixing-enhanced microchip and the multiplex surface-enhanced Raman scattering (SERS) nanotag system for direct EV phenotyping without EV enrichment. In a preclinical model, we observe the EV phenotypic heterogeneity and different phenotypic responses to the treatment. Furthermore, we successfully detect cancer-specific EV phenotypes from melanoma patient plasma. We longitudinally monitor the EV phenotypic evolution of eight melanoma patients receiving targeted therapy and find specific EV profiles involved in the development of drug resistance, reflecting the potential of EV phenotyping for monitoring treatment responses.