Mammalian cells release extracellular vesicles (EVs) into their microenvironment that travel the entire body along the stream of bodily fluids. EVs contain a wide range of biomolecules. The transported cargo varies depending on the EV origin. Knowledge of the origin and chemical composition of EVs can potentially be used as a biomarker to detect, stage, and monitor diseases. In this paper, we demonstrate the potential of EVs as a prostate cancer biomarker. A Raman optical tweezer was employed to obtain Raman signatures from four types of EV samples, which were red blood cell- and platelet-derived EVs of healthy donors and the prostate cancer cell lines- (PC3 and LNCaP) derived EVs. EVs' Raman spectra could be clearly separated/classified into distinct groups using principal component analysis (PCA) which permits the discrimination of the investigated EV subtypes. These findings may provide new methodology to detect and monitor early stage cancer.