Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa‐derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time‐resolved fluorescence immunoassay (TR‐FIA) for capture/detection of PCa‐derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin‐coated wells. After application of exosomes, Europium‐labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR‐FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR‐FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR‐FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR‐FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease‐specific detection assays.