Objective：To establish a real-time fluorescent quantitative PCR method for determining the BK virus（BKV） level in renal transplant recipients,and to evaluate its clinical application. Methods： Plasmids containing part of BKV VP1 gene conservative region were constructed as external standards, and a TaqMan probe technique was used to establish a quantitative method for determination of BKV. Urine and peripheral blood（PB） samples from 112 renal transplant recipients were assayed for BK virus levels, and the results were compared with those of 40 healthy controls. Results： The real-time fluorescent quantitative PCR assay established in this study had good sensitivity, specificity, and reproducibility. The minimal detectable level was 8 × 10^2 copy/ml, intro-experiment variation was 0. 54 %-3.78 %, and intra-experiment variation was 0.62 %-4. 58 %. BKV was detected in 27.7% urine samples and 11.6% PB samples from patients. The median level of BKV in urine and PB were 8.2 × 10^4 copy/ml and 2.4 × 10^3 copy/ml, respectively. BKV positive rate of 40 healthy population in urine and PB samples were 2.5 % and 0 %, respectively. The positive rate and level of BKV in renal transplant recipients were both significantly higher than those in normal cohort （both P〈0.01）. The positive rate of BKV in urine samples were significantly higher than that in PB samples（P=0.02）, but the BKV load in urine samples was not related to that in PB samples. Conclusion： The real-time fluorescent quantitative PCR assay in this study is simple, reliable, and precise, which lays a foundation for future study of the relationship between BKV infection and renal graft loss