Levofloxacin (Levo), a second generation fluoroquinolone, has both clinical and environmental relevance. Therefore, the implementation of fast, robust and cost effective techniques for its monitoring is required. Here, its spectroscopic characterization and its detection in aqueous environment were carried out using surface enhanced Raman spectroscopy combined with droplet based microfluidics. The Levo molecule interacts with the silver nanoparticles via the carboxylate group and it adopts an upright or slightly tilted orientation. Furthermore, it is shown that the presence of Cl⁻ ions has a strong influence on the enhancement efficiency of the Raman signal of the target molecule. Thus, for the determination of the limit of detection (LOD) the measurements were carried out in the absence of any electrolytes. The estimated LOD is ∼0.8 μM and the linear dynamic window ranges between 1–15 μM. These results were achieved after the normalization of the SERS signal to the Raman mode at 230 cm⁻¹. This band was attributed to the ν(Ag–O) stretching and it accounts for the Levo molecules in the first layer on the Ag nanoparticles.