A single step approach to tailored nanoparticle-bioconjugates, enabling the generation of gold nanoparticles by laser ablation and their in situ conjugation with any biomolecule bearing an electron donating function (e.g., thiolated oligonucleotides) is established. The integrity of oligonucleotides after conjugation and the stability of bioconjugates in physiological media are investigated. Their size is tailorable via process parameters. This rapid and universal method may provide biochemists with various nanoparticle-bioconjugates for screening the often unpredictable structure–function relationship.