Ethionamide (ETH) is a second line antitubercular drug suffering from poor solubility in water and strong tendency to crystallize. These drawbacks were addressed by loading ETH in β-cyclodextrin (βCyD)-based carriers. The drug was incorporated in a molecular state avoiding crystallization even for long-term storage and obtaining a tenfold increased solubility up to 25 mM. The binding of ETH to polymeric βCyD nanoparticles (pβCyD NPs) was investigated in neutral aqueous medium by means of solubility phase diagrams, circular dichroism (CD) and UV–vis absorption and compared with the corresponding βCyD monomer. The binding constants and the absolute CD spectra of the drug complexes were determined by global analysis of multiwavelength data from spectroscopic titrations. The spectroscopic and photophysical properties of the complexes evidenced an alcohol-like environment for ETH included in the cavity. Additionally, ETH was found to be located not only in βCyD cavities, but also in confined microdomains inside the crosslinked NPs. This double modality of complexation together with a slightly higher binding constant makes the utilization of pβCyD NPs preferable over the monomeric βCyDs. In order to pave the way to future in vitro experiments, fluorescein labeled pβCyDs were synthesized. Interestingly the FITC labeling did not hamper the encapsulation of ETH and the drug improved the fluorescent signal of FITC molecules. The βCyD-based carriers appeared as versatile “green” systems for efficient incorporation and future delivery of ETH.