This study investigates the factors influencing DNA loading on gold nanoparticles of various sizes (15, 30, 50, 80, 150, and 250 nm). The effects of salt concentration, spacer composition, and sonication on DNA loading were evaluated. Maximum DNA loading was achieved by aging nanoparticles in 0.7 M NaCl with DNA containing a poly(ethylene glycol) (PEG) spacer. Additionally, sonication during the loading process significantly increased DNA loading. Larger nanoparticles (up to 250 nm) showed DNA loading two orders of magnitude higher than smaller nanoparticles (13–30 nm) due to their larger surface area. The use of PEG spacers and sonication were found to be critical for achieving high DNA loading. The study also demonstrated that DNA loading can be further increased by sonicating the DNA/nanoparticle solution during the salt aging process. These findings are important for applications in biodetection, nanotherapeutics, and nanoscale assembly strategies. The results show that DNA loading can be tailored by adjusting nanoparticle size, salt concentration, and spacer type, providing a guideline for optimizing DNA loading on gold nanoparticles.This study investigates the factors influencing DNA loading on gold nanoparticles of various sizes (15, 30, 50, 80, 150, and 250 nm). The effects of salt concentration, spacer composition, and sonication on DNA loading were evaluated. Maximum DNA loading was achieved by aging nanoparticles in 0.7 M NaCl with DNA containing a poly(ethylene glycol) (PEG) spacer. Additionally, sonication during the loading process significantly increased DNA loading. Larger nanoparticles (up to 250 nm) showed DNA loading two orders of magnitude higher than smaller nanoparticles (13–30 nm) due to their larger surface area. The use of PEG spacers and sonication were found to be critical for achieving high DNA loading. The study also demonstrated that DNA loading can be further increased by sonicating the DNA/nanoparticle solution during the salt aging process. These findings are important for applications in biodetection, nanotherapeutics, and nanoscale assembly strategies. The results show that DNA loading can be tailored by adjusting nanoparticle size, salt concentration, and spacer type, providing a guideline for optimizing DNA loading on gold nanoparticles.