Ultrashort pulsed laser ablation in liquids represents a powerful tool for the generation of pure gold nanoparticles (AuNPs) avoiding chemical precursors and thereby making them especially interesting for biomedical applications. However, because of their electron accepting properties, laser-generated AuNPs might affect biochemical properties of biomolecules, which often adsorb onto the nanoparticles. We investigated possible effects of such laser-generated AuNPs on biological functionality of DNA molecules. We tested four differently sized and positively charged AuNPs by incubating them with recombinant eGFP-C1-HMGB1 DNA expression plasmids that code for eGFP fusion proteins and contain the canine architectural transcription factor HMGB1. We were able to show that successfully transfected mammalian cells are still able to synthesize and process the fusion proteins. Our observations revealed that incubation of AuNP with the plasmid DNA encoding the recombinant canine HMGB1 neither prevented the mediated uptake of the vector through the plasma membrane in presence of a transfection reagent nor had any effect on the transport of the synthesized fusion proteins to the nuclei. Biological activity of the recombinant GFP-HMGB1 fusion protein appears to have not been affected either, as a strong characteristic protein accumulation in the nucleus could be observed. We also discovered that transfection efficiencies depend on the size of AuNP. In conclusion, our data indicate that laser-generated AuNPs present a good alternative to chemically synthesized nanoparticles for use in biomedical applications.