Protein profiling of extracellular vesicles (EVs) provides important information in both clinical cancer diagnosis and relevant biological research studies. Although a variety of bioanalytical techniques have been investigated for EV characterization, limitations such as time-consuming operations, the requirement of large sample volume and demand for specialized instruments hinder their practical applications. Here, we report a simple and wash-free homogeneous colorimetric assay for sensitive detection of surface proteins on EVs. Au nanoparticles were modified with thiolated aptamers to fabricate aptasensors and incubated with EVs. Upon addition of a Au growth reagent, the solution color changed from light red to blue in the presence of target proteins and became deep red when the targets were absent. Expression of CD63, epithelial cell adhesion molecules (EpCAM), and mucin1 in EVs derived from two breast cancer cell lines (MCF-7 and MDA-MB-231) were compared, showing results consistent with western blotting results. The colorimetric assay achieves a limit of detection (LOD) down to 0.7 ng μL-1 against MCF-7 EVs based on the assessment of EpCAM expression, suggesting its potential to be applied in clinical breast cancer diagnosis.