Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B‐cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19⁺ EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA‐A,B,C, HLA‐DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age‐ and gender‐matched healthy individuals.