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, Sinopodophyllum emodi. 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.