A novel highly efficient and selective puerarin (PR) enrichment procedure based on the use of molecularly imprinted polymer (MIP) as solid-phase extraction (SPE) materials has been developed and evaluated. In the present study, two widely used functional monomers acrylamide (AA) and methacrylic acid (MAA) were compared theoretically and experimentally as the candidates for MIP preparation, showing that AA was a better functional monomer to prepare MIP. Spectroscopic analysis in conjunction with molecular modeling approach was used to study the interactions between PR and AA. Afterwards, the binding capacity and selectivity of MIP was assessed by equilibrium rebinding experiments and competitive adsorption experiments. The resulting MIP was applied to the selective extraction of PR from a real plant sample. After optimization of SPE protocol, the recovery yield of PR was up to 86% by one-step extraction from the extract of Radix Puerariae, and the purity of PR was larger than 96% by HPLC analysis. These results reported herein provide the possibility for the highly selective separation and purification of active PR from Radix Puerariae by molecularly imprinted solid-phase extraction (MISPE). (C) 2009 Elsevier B.V. All rights reserved.