Fast ex situ functionalization of gold nanoparticles with fluorophore-labeled cell-penetrating peptides is investigated with a novel liquid flow cascade injection system. Successful conjugation is proved by various methods, such as UV−vis spectrometry and electron microscopy, whereas nanoparticle size-quenching is clearly observed. By variation of the peptide concentration introduced promptly after particle generation, gold nanoparticle bioconjugates with different degrees of cluster formation and/or aggregation and different peptide surface coverage values are obtained. The sizes of synthesized inorganic−organic gold nanoparticle bioconjugate show obvious correlation with time-delayed conjugation, giving evidence that laser-generated nanoparticles continue growing outside the cavitation bubble in the multisecond time scale until achieving their final size. Introducing 6.6 μM bioactive ligands, the highest conjugation efficiency of 93\% and ζ potential of 27.5 mV is reached at the shortest delay time (200 ms), resulting in 20 nm average sized bioconjugates. Finally, in a preliminary biological application, laser scanning confocal microscopy clearly revealed an amplified cellular uptake using HIV-1 transactivator peptide-conjugated gold nanoparticles compared with nonconjugated entities within embryonic fibroblasts after a short coincubation time of 1 h. The generation of high amounts of highly pure cell-penetrating nanomarkers by the nanosecond laser-assisted fast ex situ conjugation is thus a promising method to probe biological activities such as nanodrug internalization mechanisms.