Tumor cell–derived extracellular vesicles (EVs) are being explored as circulating biomarkers, but it is unclear whether bulk measurements will allow early cancer detection. We hypothesized that a single-EV analysis (sEVA) technique could potentially improve diagnostic accuracy. Using pancreatic cancer (PDAC), we analyzed the composition of putative cancer markers in 11 model lines. In parental PDAC cells positive for KRAS ^mut and/or P53 ^mut proteins, only ~40% of EVs were also positive. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRAS ^mut and P53 ^mut protein was detectable at much lower levels, generally in <0.1% of vesicles. These vesicles were detectable by the new sEVA approach in 15 of the 16 patients. Using a modeling approach, we estimate that the current PDAC detection limit is at ~0.1-cm ³ tumor volume, below clinical imaging capabilities. These findings establish the potential for sEVA for early cancer detection.