In this work, the adsorption and orientation on gold nanoparticles (AuNps) of a new family of cruciform systems consisting of thiophene rings and imino groups were studied. The structural modification and its influence on the adsorbate‐substrate interaction were evaluated by UV–Vis spectroscopy and Surface Enhanced Raman Spectroscopy (SERS). The absence of SERS spectrum for (N,N′‐bis(4‐(trifluoromethyl)benzylidene)‐2,5‐di (thiophene‐2‐yl)‐1,4‐diaminobenzene) CFF shows that the inclusion of a trifluoromethyl group (‐CF3) on the benzylidene fragment limits the interaction of the CFF system with the gold substrate, in contrast, to that obtained for (N, N′‐dibenzylidene‐2,5‐di (thiophene‐2‐yl) ‐1,4‐diaminobenzene) 2‐CF and (N, N′‐bis (4‐methoxybenzylidene) ‐2,5‐di (thiophene‐2yl) ‐1,4‐diaminobenzene) CMF, where the adsorption took place preferentially through the thiophene rings, resulting in partial quinoidization. On the other hand, the interaction for compound (N, N′‐bis (4‐methylenepyridinyl) ‐2,5‐di (thiophene‐2‐yl) ‐1,4‐diaminobenzene) CPy with the surface was conducted by means of the pyridinic fragments. The systematic modification of the bifunctional cruciform systems, with groups of different nature, makes it possible to rationalize the structural aspects that directly influence the adsorbate‐substrate interaction and molecular orientation on gold substrates. These structural parameters are the basis to the development of stable molecular assemblies, which can act as basic building blocks in the manufacture of molecular switches. The adsorption and orientation of a new family of cruciform systems were evaluated by Surface Amplified Raman Spectroscopy (SERS). In this sense, the structural modifications allowed modulating the substrate‐adsorbate interaction center, affinity, and preferential orientation of the adsorbates with respect to the gold nanoparticles.