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A selective and affinitive molecularly imprinting polymer (MIP) was prepared to podophyllotoxin (PPT) using a non-covalent imprinting approach. The imprinted conditions including the kind and relative amounts of functional and cross-linking monomers were carefully optimized. The binding characteristics of the imprinted polymer were evaluated in different solvent systems using equilibrium binding experiments. UV spectrum revealed cooperative hydrogen-bonding complex between PPT and acylamide (AM) might be formed at the ratio of 1:2 in the prepolymerized system, and produced two classes of binding sites in the resulting MIP with dissociation constants of 0.337 and 5.76 mmol l−1, and the affinity binding sites of 78.67 and 346.0 μmol g−1, respectively. Selective binding of the template molecule is demonstrated in comparison to the analogue 4′-demethylpipodophyllotoxin (DMEP). Finally, the optimized MIP was used as an adsorbent to extract PPT directly from the ethanol extraction of Chinese traditional Tibetan medicine, <b>Sinopodophyllum emodi</b>. The binding selectivity and recovery of the optimized imprinted polymer had been further estimated by HPCE and compared to its non-imprinted polymer. The obtained results implied a possibility to extract PPT directly from plants by a MIP technique shown in this paper.