The surface interactions between gold nanoparticles (AuNPs) and lipase from Candida antarctica fraction B (CALB) were investigated at macromolecular and colloidal length-scales. In order to elucidate the reciprocal effect of the interactions on the individual component's properties, CALB was either added during the synthesis of AuNPs or added to pre-synthesized AuNPs. In both cases, it was observed that the AuNPs are spherical and stable and, by fluorescence and circular dichroism spectroscopies, changes were observed in the secondary and tertiary structure of CALB, that were dependent on the AuNPs concentration. Nevertheless, the catalytic activity of CALB was maintained, although at a lower percentage (≥ 80%), thus new bio-functionalities were inserted into AuNPs upon interaction with CALB. Using simple and straightforward approaches and state-of-the-art techniques important knowledge about CALB/AuNPs bioconjugates was gained, thus contributing to the development of new nano-biomaterials and for the safe use of AuNPs in biomedical applications.