The objective of this study was to screen for antigens of the hepatitis C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23 HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8 HCV-negative sera. A total of 300 clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS 11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2 NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.