Pancreatic cancer has led to an extremely high mortality rate because of its insidious onset and lack of early clinical symptoms. Effective early diagnosis is essential to improve the treatment of pancreatic cancer. Tumor-secreted extracellular vesicles (EVs) have attracted great interest as potential tumor biomarkers. However, most of the methods for detecting serum EVs have some general problems such as cumbersome, time-consuming extraction steps, and high cost, which limit greatly the research on cancer detection based on EVs. Herein, we report a light-initiated chemiluminescent assay (LICA) method using photosensitive beads for direct detection of EVs in serum enriched with ephrin type-A receptor 2 (EphA2), which show high expression in pancreatic cancer patients. Combining with a serum biomarker CA19-9, pancreatic cancer patients could be distinguished rapidly by sensitive detection of EphA2-EVs from serum without any purification. This developed method could be extended to improve the diagnosis efficiency for other cancers and gain an insight into EV detection.