The production of nanostructured materials for biological and medical applications may be applied toward the conjugation of adequate substances to boost the stimulus response of sensors and diagnostic probes. In this sense, Langmuir–Blodgett films constituted of bioinspired and biomimetic materials have attracted attention because of the ease of manipulation of the molecular architecture. In this paper, we employed a nucleoside-based drug, which was linked with a sterol hydrophobic moiety (3′,4′-acetonide-uridine-succinate-cholesterol conjugate) to provide it an amphiphilic character. The drug was spread on the air–water interface, alone or mixed with stearic acid, forming Langmuir monolayers, and the complex Eu(tta)3(H2O)2 was incorporated in the drug-containing monolayer. Interactions at the air–water interface between stearic acid, the drug, and the europium complex were then investigated with tensiometry, surface potential, infrared spectroscopy, and Brewster angle microscopy. The Langmuir films were transferred to solid supports as Langmuir–Blodgett films, which presented luminescent properties that could be tuned according to the molecular architecture. We believe that these results can serve as a novel approach to characterize and assemble materials organized in the molecular scale for medical applications.