Fluorescent analogues of the gypsy moth sex pheromone (+)-disparlure (1) and its enantiomer (-)-disparlure (ent-1) were designed, synthesized and characterized. The fluorescently labelled analogues 6-FAM (+)-disparlure 1a 6-FAM (-)-disparlure ent-1a were prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) of disparlure alkyne and 6-FAM azide. These fluorescent disparlure analogues 1a ent-1a were used to measure the disparlure binding to two pheromone-binding proteins from the gypsy moth, LdisPBP1 and LdisPBP2. The fluorescence binding assay using 6-FAM disparlure enantiomers 1a and ent-1a showed that the LdisPBP1 and LdisPBP2 have different binding affinities with 1a and ent-1a. The LdisPBP1 has stronger affinity for 6-FAM (-)-disparlure ent-1a, whereas LdisPBP2 has stronger affinity for 6-FAM (+)-disparlure 1a, consistent with the findings from previous study with disparlure enantiomers. The 6-FAM disparlure enantiomers appeared to be much stronger ligands for LdisPBPs, with the binding constant (Kd) in nanomolar range, compared to the fluorescent reporter such as 1-NPN (which had Kd values in micromolar range). The fluorescence competitive binding assays were used to determine the displacement constant (Ki) for the disparlure enantiomers in competition with fluorescent disparlure analogues binding to LdisPBP1 and LdisPBP2. The Ki data showed that disparlure enantiomers can effectively displace the fluorescent disparlure from the binding pocket of LdisPBPs.