An important point regarding the development of stable biofunctional nanoparticles for biomedical applications is their potential for aspecific interactions with the molecules of the biological environment. Here we report a new self-assembled ligand monolayer system for gold nanoparticles called Mix-matrices, formed by a mixture of HS-PEG and alcohol peptides (peptidols) molecules. Stability of the Mix-capped nanoparticles prepared in various conditions was assessed using tests of increasing stringency. The results highlight the importance of identifying a concentration of ligands sufficiently high to obtain a compact matrix when preparing nanoparticles and that the stability of capped nanoparticles in biological environments cannot be predicted solely on their resistance to electrolyte-induced aggregation. The Mix-capped nanoparticles are resistant to aggregation induced by electrolytes and to aspecific interactions with proteins and ligand exchange. In addition, Mix-matrices allow the easy introduction of a single recognition function per nanoparticle, allowing the specific and stoichiometric labeling of proteins with gold nanoparticles. Therefore, the Mix-matrices provide a useful tool for the development of nanoparticle-based quantitative bioanalytical and imaging techniques, as well as for therapeutic purposes, such as the specific targeting of cancerous cells for photothermal destruction.