A new molecularly imprinted fluorescent chemosensor with selectivity for spermidine has been developed using quinoline modified vinyl-β-cyclodextrin as a reporter and major functional monomer. Acrylamide that provided synergetic monomer/template interactions was also used in the imprinting. The imprinted receptor of the chemosensor was created by the inclusion and hydrogen-bonding interactions between functional monomers and spermidine. The chemosensor has shown higher sensitivity and imprinting performance than that synthesized using only β-cyclodextrin. The imprinting factor of the imprinted membrane for spermidine is 3.87. This suggests that the imprinted chemosensor has good imprinted binding affinity. The selectivity (α), evaluated by the ratio of sensitivity for spermidine to that for its analogs, was ≧2.0, indicating the suitable recognition properties of the chemosensor. The fluorescence of the imprinted membrane exhibited a “Turn-on” response mode. A linear relationship between the negative logarithm of spermidine concentration and the fluorescence intensity of the chemosensor at C_spermidine of 5 × 10⁻⁷ to 1 × 10⁻⁴ mol L⁻¹ has been obtained. The results demonstrated that the use of vinyl-β-cyclodextrin and radical polymerization for this chemosensor preparation has obvious advantages. The imprinted fluorescent chemosensor has application potential for spermidine determination in complex samples.