Enolase (EC 188.8.131.52), also called 2-phospho-D-glycerate hydrolase or 2-phosphoglycerate dehydratase, is a key enzyme in glycolysis. It converts 2-phosphoglycerate to phosphoenolpyruvate (PEP) & also catalyzes the reverse reaction, PEP to 2-phosphoglycerate under anabolic conditions during gluconeogenesis. This enzyme exists in all organisms, which can undergo glycolysis. Enolase activity is easily detectable in extracellular vesicles (EVs) derived from eukaryotic cells and it could be used for evaluating functionality and stability of EVs. Moreover, it’s increased activity is associated with tumorigenesis and therefore precise measurement of enolase activity may be of great interest for EV-based tumor diagnosis.