A new fluorescent-cholesterol (Cum-Chl) molecule has been synthesized by attaching 3-acetyl-7-(diethylamino)-2H-chromen-2-one (Cum) to cholesterol via cholesterol bound β-keto ester. The β-keto ester was synthesized from the corresponding nitrile by applying the Blaise reaction. The molecule forms H-aggregates in solutions. The efficiency of aggregation is high in water. The H-aggregates are non-fluorescent in non-aqueous solvents but fluorescent in water. Aqueous bile salt media suppress the formation of H-aggregates, this effect being more pronounced for sodium deoxycholate (NaDC) which is more hydrophobic. With increasing bile salt concentration, the enhancement of monomeric fluorescence intensity of Cum-Chl generally follows the progressive miceller aggregation of bile salts. Incorporation of Cum-Chl into the dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane results in a significant enhancement of monomeric fluorescence intensity. The variation of fluorescence intensity is also sensitive to the thermotropic phase transition of the bilayer. It is seen that in such aqueous micro- and nanoscale organized media like bile salts and lipid bilayer membranes the monomer-to-aggregate fluorescence intensity ratio reflects the state of organization of the medium.