Abstract： Objective：To prepare the national secondary reference material of HCV RNA for nucleic acid amplification technology. Methods：The candidate sample of HCV RNA positive plasma was diluted to concentration of approximately 3.0×10⁵ IU/ml with HCV negative human plasma and then lyophilized and dispensed as 1 mL for each tube. The homogeneity and stability of the prepared reference materials were tested and the clinical applicability was evaluated. The quantity of prepared material was traced to the international standard (06/102). Results:The evaluation of homogeneity indicated the between run precision was 1.77% and the within run precision was 1.40% (F=1.596 9, P>0.05). The prepared materials could be stable at room temperature (20-25 ℃) for 14 days, at 2-8 ℃ for 2 months and at 20 ℃ for 12 months (P>0.05), but was unstable at 37 ℃(P<0.05). Under the conditions of opening the tube after 1 week, repeatedly thawing/freezing for 5 cycles and simulated transportation between two laboratories, no significant differences were observed in the comparisons of the test results of the prepared materials with controls stored at -20 ℃(P>0.05). The quantity of the prepared material of HCV RNA was determined as (1.9±0.9)×10⁵ IU/mL. Conclusion: The prepared material of HCV RNA reached the requirements of the national secondary reference and can be used as the standard for nucleic acid amplification test of HCV RNA.