The reaction of polyurethane prepolymers with fluorescein (free acid) and glycerin or castor oil-cross-linker has been performed in N,N-dimethylformamide and in such way a cross-linked elastomer material has been obtained. Polyurethane prepolymers were prepared by reaction of the poly(1,4-butane) diols (Terathane 1400) with aliphatic 1,6 hexamethylene diisocyanate. The above polymerization reactions lead to the formation of novel aromatic-aliphatic polyurethane elastomers having fluorescein moieties in the main chain. The resulting novel cross-linked polyurethanes have excellent mechanical properties, especially in the castor oil polyurethane elastomer formulations and show light-violet fluorescence property and could be used as photoactive polymers for labeling materials. These polyurethanes were characterized through IR, TGA, DSC, and mechanical analyses. Some aspects of the fluorescent emission in fluorescein-polyurethane elastomers were studied.