Extracellular vesicles (EVs) function in intercellular communication and can be indicative of the biological status of their producing cells, for example in disease. Isolation of EVs from blood or tissue culture medium is instrumental for the discovery of novel EV associated disease biomarkers as well as for functional studies. Current protocols for EV isolation from blood are limited by low recovery yield and in their capacity to remove contaminating particles, including lipoprotein particles and protein aggregates. In this study, we designed and evaluated a three step protocol to isolate EVs from blood or tissue culture media, with high yield and purity. EVs were first collected and separated from the majority of soluble plasma protein by precipitation with polyethylene glycol (PEG). PEG precipitation resulted in near to absolute recovery, while the yield of EVs collected by ultracentrifugation was only 15%. PEG precipitated EVs were then separated from co-precipitated proteins and protein complexes, low density lipoproteins (LDL), and the majority of high density lipoproteins (HDL) by upward displacement into a iohexol density gradients, yielding 493 fold purification relative to total plasma protein with 76% recovery. Finally, remaining HDL and iohexol was effectively removed by size exclusion chromatography (SEC). Near to absolute pure of isolated EVs was confirmed by cryo-electron microscopy.