In this paper, in situ and ex situ approaches were used for the preparation of gold nanoparticles-bovine serum albumin bioconjugates. The comparison between the binding constants (K-b) and Gibbs free energies obtained from the fluorescence spectra of both approaches reveals that the Kb increases, while the Gibbs free energy decreases by decreasing the size of AuNPs in the in situ generation of the AuNPs-BSA bioconjugated system, which indicates a greater interaction of smaller AuNPs with BSA. We also showed that the use of pulsed-laser ablation with a wavelength of 1064 nm has no effect on the photo degradation of BSA. The in situ bioconjugation of AuNPs with BSA produces ultrasmall nanoparticles in comparison to the ex situ bioconjugation. In addition, the ultrasmall nanoparticles have different interaction with BSA in comparison to the larger AuNPs. Due to the importance of gold nanoclusters, theoretical calculations were performed to dock an Au-42 nanocluster into BSA. The results show that the nanoclusters enter into the BSA and strongly interact with it, rather than the interaction with the BSA surface. Also, the best binding sites inside the BSA structure for the interaction with Au-42 were determined using docking calculations. (C) 2016 Elsevier B.V. All rights reserved.