Most cells secrete vesicles into the extracellular environment to interact with other cells. These extracellular vesicles (EVs), have undergone a paradigm shift upon the discovery that they also transport important material including proteins, lipids and nucleic acids. As natural cargo carriers, EVs are not recognised by the immune system as foreign substances, and consequently evade removal by immune cells. These intrinsic biological properties of EVs have led to further research on utilising EVs as potential diagnostic biomarkers and drug delivery systems (DDSs). However, the internalisation of EVs by target cells is still not fully understood. Moreover, it is unclear whether EVs can cross certain biological membranes like the blood-brain barrier (BBB) naturally, or require genetic modifications to do so. Hence, this review aims to evaluate the relationship between the composition of EVs and their association with different biological membranes they encounter before successfully releasing their cargo into target cells. This review identifies specific biomarkers detected in various EVs and important biological barriers present in the gastrointestinal, placental, immunological, neurological, lymphatic, pulmonary, renal and intracellular environments, and provides a recommendation on how to engineer EVs as potential drug carriers based on key proteins and lipids involved in crossing these barriers.