The synthesis of a series of β-cyclodextrin (CD) dimers in which the two macrocycle units are linked by rigid-rod phenylene-ethynylene tethers of different length and shape through the cyclodextrin secondary sides is reported. The synthesis involves Sonogashira coupling reactions for construction of the tethers and final grafting of the CD units by click chemistry. The self-aggregation of the synthesized CD dimers in aqueous medium was investigated by fluorescence spectroscopy, isothermal titration calorimetry and dynamic light scattering. Rigid-rod-tethered cyclodextrin dimers formed polydisperse nanoparticles of 240 to 480 nm in water solution depending on the tether shape, and exhibited fluorescence. The binding and fluorescence sensing abilities of the CD dimers toward sodium cholate and deoxycholate were studied. As a result, the newly synthesized rigid-rod-tethered cyclodextrin dimers proved to be very sensitive fluorescent chemosensors. A new cyclodextrin-based ratiometric fluorescence probe has thus been developed for the detection of bile salts such as sodium cholate and deoxycholate.