Agglomeration/aggregation is an indispensable phenomenon observed by different nanoparticles. In the present study, commercial grade (50 nm) and chemically synthesized (40 nm) gold nanoparticles (AuNPs) were aggregated at sub-zero temperatures, followed by disruption of the AuNP aggregates via nanosecond laser-ablation and subsequent effect on biological interactions. AuNPs were characterized pre/post laser-ablation via UV-visible spectroscopy, transmission electron microscopy, atomic force microscopy, etc. The process of freezing (aggregation) and laser-ablation (dispersion) was performed multiple times, in order to compare the yield of nanoparticles after each cycle of laser-ablation. Further, AuNPs pre/post laser-ablation were assessed for cytotoxicity, protein-corona formation, and cell-uptake by in vitro studies using RAW264.7, Caco-2 and Neuro-2 a cell lines. Aggregates for both the types of AuNPs displayed fragmentation following first cycle of laser-ablation. In addition, AuNPs obtained after fragmentation of the aggregates showed reduction in diameter and reshaping, as compared to native AuNPs. The size and shape of the nanoparticles after second and third cycle of laser-ablation was same as that obtained after first cycle of ablation. Both laser-ablated and native AuNPs showed similar effects on viability of RAW 264.7 and Caco-2 cells, after 24 h and 48 h of exposure. Cell uptake of native and laser-ablated AuNPs was observed to be a size dependent phenomenon. Present findings showed that nanosecond laser ablation of cryoaggregated AuNPs lead to changes in the physical properties of AuNPs post ablation like size and shape, however, biological interaction with cells remained same. This work is first report on biological interactions of AuNPs generated via laser-ablation of cryoaggregated AuNPs.