Exosomes are cell‐derived vesicles secreted by both normal and cancerous cells into the extracellular matrix and in blood circulation. Tumor‐derived exosomes have attracted increasing attention in noninvasive cancer diagnosis and prognosis. However, their effective capture and specific detection pose significant technical challenges. Current detection methods largely fail to quantify the tumor‐derived exosomes present in the total (bulk) exosome population derived from body fluids of cancer patients. In this proof‐of‐concept study, we report an electrochemical detection method to directly quantify the disease‐specific exosomes present in cell culture media. The assay has a two‐step design, where bulk exosome populations are initially captured by using a generic antibody (i.e. tetraspanin biomarker, CD9). Subsequent detection of the cancer‐specific exosomes within the captured exosomes was carried out by using a cancer‐specific antibody, in this case, a human epidermal growth factor receptor 2 (HER‐2) antibody, allowing quantification of HER2‐postive, breast‐cancer‐derived exosomes. This approach exhibits excellent specificity for HER‐2(+) BT‐474 cell‐derived exosomes (detection limit, 4.7×105 exosomes μL−1) with a relative standard deviation of <4.9 % (n=3). We suggest that this simple and inexpensive electrochemical method could be an alternative for the quantification of exosome subpopulations in specific disease settings for future clinical bioassays.