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Despite widespread storage of frozen whole blood (FWB) in biobanks worldwide, its suitability for extracellular vesicle (EV) research remains largely unassessed. Here, we developed a robust and practical workflow for EV-based cancer biomarker analysis from FWB by comparing differential ultracentrifugation (dUC), size-exclusion chromatography, and their combination. All methods successfully recovered vesicles within the expected 50-150 nm range, enriched for canonical EV markers, while reducing blood-derived EV contaminants, and displayed vesicle-like morphology. Among tested methods, dUC emerged as the most cost-effective and labor-efficient approach. Applying this pipeline to biobanked FWB samples from patients with small intestinal neuroendocrine neoplasms yielded an average of ∼1,000 proteins detected per sample using mass spectrometry-based proteomics, including known neuroendocrine markers and enrichment in synapse organization signaling pathways. These findings demonstrate the feasibility of EV isolation from FWB and biomarker-relevant downstream analysis, highlighting the untapped potential of EV-based biomarker discovery across diseases from existing biobanks.