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.magnified image