The study investigates the factors influencing DNA coverage on gold nanoparticles, focusing on salt concentration, spacer composition, nanoparticle size, and sonication. Maximum DNA loading was achieved by aging nanoparticles in a salt solution (~0.7 M NaCl) containing DNA with a PEG spacer. Sonication during the surface loading process significantly increased DNA loading. Larger nanoparticles (up to 250 nm) showed a two-orders-of-magnitude higher DNA loading compared to smaller (13–30 nm) nanoparticles due to their larger surface area. The findings highlight the importance of these parameters in optimizing DNA loading for biodetection and nanotherapeutic applications.The study investigates the factors influencing DNA coverage on gold nanoparticles, focusing on salt concentration, spacer composition, nanoparticle size, and sonication. Maximum DNA loading was achieved by aging nanoparticles in a salt solution (~0.7 M NaCl) containing DNA with a PEG spacer. Sonication during the surface loading process significantly increased DNA loading. Larger nanoparticles (up to 250 nm) showed a two-orders-of-magnitude higher DNA loading compared to smaller (13–30 nm) nanoparticles due to their larger surface area. The findings highlight the importance of these parameters in optimizing DNA loading for biodetection and nanotherapeutic applications.