4,4′-diaminostilbene (DAS) was used as a molecular linker of Ag nanoparticles prepared by reduction of silver nitrate by sodium borohydride. The influence of DAS concentration on the nanoparticles aggregation was investigated by Surface enhanced Raman scattering (SERS), UV–vis spectroscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). It is revealed that linking Ag nanoparticles with hot spots formation occurs only at sub- or monolayer surface coverage up to 5 × 10⁻⁶ mol/l. At multilayer coverage, only single-end interaction of molecules with a surface is possible. The effects of pH and halide salts on the adsorption mechanism of DAS were also checked. The protonation of amino groups and presence of chloride anions on a surface prevent the linkage of Ag nanoparticles. As a result, a simple and versatile method for obtaining Ag nanoparticle aggregates of controllable size and optical properties is proposed. The corresponding limitations are described for a potential application of the developed hot spots substrates.