Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq™ antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using live cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.