Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.