Blood plays a vital role in diagnostic medicine by providing essential information for assessing health and diagnosing various conditions. Routine blood tests can reveal important biomarkers that support early diagnosis and timely intervention. Recently, extracellular vesicles (EVs) have emerged as promising biomarkers due to their role in intercellular communication and involvement in disease processes such as cancer and neurodegeneration. However, isolating EVs from plasma remains challenging, as residual blood cells, especially platelets, can interfere with vesicle release. In this study, we explore the use of the PRotein Organic Solvent PRecipitation (PROSPR) method, a straightforward and effective technique previously applied to plasma and brain samples, to isolate EVs directly from whole blood. We performed detailed analyses, including ultrastructural imaging, cytometry, and multi-omics profiling, to characterize the EVs obtained. Our findings demonstrate that PROSPR can successfully capture even the smallest exosomes (30–120 nm), which are often missed in plasma-based isolation. This approach represents a significant advancement in EV research and may enhance the identification of blood-based biomarkers for a range of diseases.