Nanobodies (Nbs) are single-domain antibodies with highly desirable properties employed as versatile tools in structural biology, biochemistry and biomedical sciences. Nbs also have a significant potential for the development of highly specific and user-friendly diagnostic assays. These immuno-assays often employ gold nanoparticles (AuNPs) as a detection label. Despite great advances in Nb technology and the immuno-sensor field, the functionalization of AuNPs with Nbs mostly remains unchartered territory. In this paper, we present a study of the functionalization of AuNPs with Nbs through physical adsorption. We show that Nb pI, solution pH, additives and protein engineering strategies have profound effects on the generation of stable Nb-AuNPs. Our results further demonstrate that physical adsorption can be used to produce functional Nb-AuNPs that could be employed in a wide array of applications.