Analysis of multiple surface proteins on extracellular vesicles (EVs) can reveal biological characteristics and potential therapeutic targets of cancer, particularly in highly heterogeneous breast cancer. However, due to the limited surface area of EVs, spatial hindrance remains a challenge for multiprotein assessment. Here, we present a sequential labeling-assisted electrochemical method for the precise and multitarget analysis of surface proteins on EVs, using breast cancer-related epidermal growth factor receptor and programmed death ligand-1 as examples. This sequential labeling is achieved through the use of a pair of aptamer probes functionalized with electroactive nanoparticles and an oxidative cleavage process facilitated by the bleomycin-Fe