Trace concentrations of polycyclic aromatic hydrocarbons (PAHs) have been successfully detected using surface-enhanced Raman scattering (SERS) spectroscopy. For such purpose, new SERS substrates have been developed, consisting of Ag nanoparticles, either in suspension or immobilized on glass, obtained by a new method and covered by adsorbed self-assembled calix[4]arene molecules. Among the assayed calix[4]arenes, the 25,27-dicarboethoxy-26,28-dihydroxy-p-tert-butylcalix[4]arene host molecule displays analytical selectivity to the PAH guest systems bearing four benzene rings, mainly pyrene. The host-guest interaction mechanism seems to take place through a pi-pi stacking interaction leading to a charge transfer between the complex and the metallic surface, which may also induce a notable influence on the surface charge of the metallic nanoparticle.