Gold nanoparticles (AuNPs) have been reported to facilitate double-stranded DNA dissociation and improve performance of several PCR systems. Here we investigated AuNPs' effect on GC-rich DNA amplification. We found that AuNPs could enhance PCR amplification of the GNAS1 promoter region (∼84% GC) mediated by Pfu or Taq DNA polymerase. However, under optimal concentrations of AuNPs, higher amounts of Taq were required. Furthermore, the GC-rich FMR1 (80.4% GC) gene of Homo sapiens as well as exoT (67.3% GC), exsE (71% GC) and pqqF genes (74% GC) of Pseudomonas aeruginosa were also efficiently amplified. AuNPs can become an effective additive in GC-rich PCR and facilitate analysis of challenging genomic sequence in basic and clinical research.