Objective To establish a fluorescence quantitative-reverse transcription-polymerase chain reaction (FQ-RT-PCR) with two-probe for quantification of HCV RNA loads,and to optimize the experimental conditions to increase sensitivity and specificity.Methods HCV RNA loads in 89 cases with positive anti-HCV and 220 cases with negative anti-HCV were quantified by FQ-RT-PCR with double probe,and the results were compared with another two commercial HCV RNA quantificationkits simultaneously.Results For the 2 groups (89 cases with positive anti-HCV and 220 cases with negative anti-HCV), the positive rate of HCV RNA was 91.0%(81 cases) and was 2.27%(5 cases) respectively by FQ-RT-PCR with two-probe, while it was 82.0% (73 cases)and 0.45% (1 cases)), 79.7%(71 cases))and 1.81%(4 cases) respectively by using the two commercial kits.Conclusion FQ-RT-PCR assay with two-probe may increases the sensitivity and specificity for the detection of HCV RNA loads comparing with commercial kits.