Objective To develop an effective preparation method to improve the sensitivity and accuracy of real-time PCR in detection of BK virus’s (BKV’s) load in urine samples. Methods A total of 24 samples documented as positive probes in primary detection were enrolled in this study. The candidate samples were prepared by 4 different approaches: unprocessed urine, BKV’s DNA extracted from urine, 110 diluted urine, and 1100 diluted urine; and then they were subjected to real-time PCR examination to obtain the viral load. The data obtained were analyzed by SPSS 11.0. Results The four different preparation processes for urinary specimens had significant impact on detection results of real-time PCR. Three samples were negative in the unprocessed urine group and 66.7% of its samples had the lowest viral loads compared with the other three groups. Two samples in the 1100 diluted urine group were negative and 79.2% of its samples had the highest viral loads, but its median load was similar to that of the 110 group. Viral gene was detected in all samples in the DNA extraction group and 110 diluted urine group, but the loss of the target gene was more severe in the DNA extraction group. Conclusion The 110 diluted urine is better for real-time PCR detection of BKV’s load, as it lose less viral gene and is more efficient, easy to perform and economical.