Femtosecond-pulsed laser ablation of gold is investigated in electron donor moiety containing biomolecule solutions, resulting in the controlled design of biocompatible, partially charged and electrostatic-stabilized gold nanoconjugates by in situ surface functionalization. The cell-penetrating peptide penetration and a targeting mini Strepaptamer sequence are conjugated simultaneously to a single nanoparticle for the first time, producing highly stable gold nanoparticle bi-conjugates with defined degrees of surface coverages for each biomolecule. Ablating gold by 100 μJ laser pulses in presence of 1 μM penetration and 4.5 μM mini Strep yields an average bioconjugate size of 10nm and results in 61pmol cm-2surface coverage for the aptamer and 30 pmol cm-2 for the penetration. Whereat, degree of surface coverage shows direct correlation to process parameters as well as to biomolecule size, concentration and steric dimension of the biomolecules used. To investigate the influence of time-delayed bioconjugation in view of more efficient up-scaling process, a fast ex situ conjugation is presented in a continuous flow chamber secondary, allowing highly controlled bioconjugation without irradiating the photo-and thermal-sensitive biomolecules and permitting the use of nanosecond laser pulses.