A gold nanoparticle probe-based assay (GNPA) was developed for ultrasensitive detection of Hepatitis C virus (HCV) core antigen. In the GNPA, after anti-HCV core antigen polyclonal antibodies and single stranded barcode signal DNA were labeled on gold nanoparticle probe (NP), DNA enzyme was used to degrade the unbound barcode DNA5. The anti-HCV core antigen monoclonal antibodies were coated on magnetic microparticles probe (MMP). Then the NP-HCV core antigen-MMP sandwich immunocomplex was formed when the target antigen protein was added and captured. Magnetically separated, the immuno-complex containing the single-stranded barcode signal DNA was characterized by TagMan probe based real-time fluorescence PCR. A detection limit of 1 fg/ml was determined for the HCV core antigen which is magnitude greater than that of ELISA (2 ng/ml). The coefficients of variation (CV) of intra-assay and inter-assay respectively ranged from 0.22-2.62% and 1.92-3.01%. The improved GNPA decreased the interference of unbound barcode DNAs and may be an new way for HCV core antigen detection. (C) 2017 Published by Elsevier B.V.