Abstract: Synthetic biotin-binding polymers were prepared by molecular imprinting. Methacrylic acid (MAA) was copolymerized with ethylene glycol dimethacrylate in the presence of biotin methyl ester (B-Me) in chloroform. Hydrogen-bonding-based complexation of B-Me with MAA generates the binding sites complementary to B-Me after extracting B-Me from the resulting copolymers, Data from NMR titration suggest a one-to-one prepolymerization complex formation of B-Me with MAA in chloroform. A possible complex structure was estimated by docking of the most stable conformers by intermolecular Monte Carlo conformational search under the assumption of a one-to-one association. The selectivity of the imprinted polymers was investigated and an imprinted polymer-based competitive binding assay for B-Me was demonstrated using biotin p-nitrophenyl ester as a nonisotopic- labeled ligand