Pandemics caused by viruses have resulted in incalculable losses to human beings, which are exacerbated due to the lack of antiviral drugs. Sulfonic group modified nanomedicine has been proved to possess a broad-spectrum antiviral ability. However, it is very challenging to maintain the antiviral activity in a high protein environment in vivo. To improve the tolerance to the complex biological environment, sulfonic mixed-charge modified gold nanoparticles (MC_AuNPs) were prepared in this research by introducing positively charged ligands into sulfonic ligand modified gold nanoparticles. The MC_AuNPs showed excellent non-fouling ability while retaining comparable antiviral ability to single sulfonic ligand modified gold nanoparticles (MDS_AuNPs). The MC_AuNPs maintained their antiviral ability in 10 mg mL⁻¹ protein solutions, but the MDS_AuNPs completely lost their antiviral capability in 1 mg mL⁻¹ protein medium. The mixed-charge modification strategy provides a practical avenue to maintain the antiviral capability of HSPG mimicking nanoparticles in high protein environments.