In Korea, the incidence of vancomycin-resistant enterococci (VRE) infection has recently been increasing, with a clinical isolation rate of up to 25%. We evaluated the clinical usefulness of real-time PCR for the detection of the vanA gene over conventional culture in patients confirmed as being VRE-colonized. During the evaluation stage, 100 consecutive clinical specimens were analyzed for the presence of the vanA gene via real-time PCR, for which 4 specimen preparations were used. During the application stage, 1,115 specimens from 82 patients were monitored for 20 months by vanA real-time PCR. For isolates prepared via incubation in broth for 24 h, the PCR results were concordant with those of the conventional method. The median value of the time spent in isolation and the number of test repeats from detection to release from isolation was 71 days and 5.5, respectively. The optimal negative test number for the release from isolation was ≥3. In conclusion, the real-time PCR method is more sensitive than, and is expected to replace, the conventional VRE surveillance method.