In the present study, a highly efficient method, referred to as optimized ultrafiltration (OUF), was developed. This method is effective for exosome purification and also facilitates clinical work involving substantial urinary exosome isolation. In the OUF method, 0.22‑µm filters along with a dialysis membrane with a molecular weight cut‑off of 10,000 kDa were introduced, in order to remove extracellular microvesicles that were >200 nm and concentrate the supernatant up to 1/50 of the initial volume. The existence, purity and production of the exosomes isolated by OUF and conventional ultracentrifugation (UC) were systematically compared by transmission electron microscopy, western blotting and nanoparticle tracking analysis. In addition, colloidal Coomassie‑stained gel and reverse transcription‑quantitative polymerase chain reaction were used to investigate the stability and integrity of exosomes isolated by these two protocols. The time required and cost of these two methods in the process of isolating urinary exosomes were also estimated. The results indicated that OUF clearly outperforms UC in quantity, quality and biological stability, and this improved method may have extensive applications in the growing fields of clinical biomarker discovery and exosome research.