Extracellular vesicles (EVs) present new opportunities for cancer diagnoses and treatment monitoring as circulating biomarkers. These cell-derived membrane-bound vesicles are abundantly present in biological fluids and carry cell-specific cargos, such as lipids, proteins and genetic materials, which can be harnessed as a minimally invasive means to probe the molecular status of tumors. EV analyses, however, poses unique technical challenges due to EVs' nanometer size, heterogeneity, and presence in vast biological background. We have previously developed nanoplasmonic sensing platforms that can rapidly and sensitively detect tumor EVs directly from clinical samples. Drawing upon our previous experiences with the prototypes, the goal of this application is to further advance the cutting-edge nanoplasmonics technology for comprehensive molecular analyses at single EV resolution. Specifically, we will 1) adapt gold nanorod structures that provides higher sensitivity down to a single molecular level and 2) integrate plasmon-enhanced fluorescence detection for multiplexed EV protein and RNA analyses in single EVs. We hypothesize that the approach will be more sensitive and comprehensive in profiling EV protein and mRNA markers than is currently possible. Using an ovarian cancer as a model system, we will evaluate 1) system implementation for high-throughput single EV detection; 2) reliable detection of tumor-derived EVs in clinical samples; and 3) multiplexed EV marker screening on tumor-derived EVs. If successful, the developed platform would render a more accessible tool to significantly accelerate the clinical adoption of EV analyses as routine screening tests for cancer care in clinical settings.