A fluorescent polyrotaxane (PR) made from the diazido-poly(ethylene glycol) (N3-PEG-N3) axis, α-cyclodextrins, and alkyne-functionalized rhodamine B (RhB-alk) stoppers via Cu(I)-catalyzed azide–alkyne click chemistry is reported. A nanowire-like morphology of the prepared fluorescent PRs was visualized by atomic force microscopy. The fluorescence emission intensities of RhB-capped PEG (RhB-PEG-RhB) and the PR were much higher than that of neat RhB or RhB-alk with the same concentration of RhB fluorophore in both dimethylsulfoxide and alkaline aqueous solutions. Fluorescence lifetimes were detected as 3.59, 3.31, 2.99, and 2.99 ns for neat RhB, RhB-alk, RhB-PEG-RhB, and the PR, respectively. The PRs with unique fluorescence properties might have further applications in the fields of biomedicine and bionanotechnology.