Non-covalent and covalent imprinting strategies have been investigated for the synthesis of stigmasterolimprinted polymers. The synthesized molecularly imprinted polymers (MIPs) were then evaluated fortheir recognition and selectivity towards stigmasterol via static and dynamic batch-binding assays andtheir performance measured against control non-imprinted polymers (NIPs). MIPs prepared using theconventional non-covalent imprinting method displayed little to no binding affinity for stigmasterolunder various conditions. In contrast, the application of a covalent imprinting approach using the novelpost-synthetically cleavable monomer-template composite stigmasteryl-3-O-methacrylate resulted inthe fabrication of a MIP that successfully recognized stigmasterol in both organic and partially aqueousenvironments. The affinity and selectivity of the covalently prepared MIP was enhanced when undertakenin a partially aqueous environment consisting of an acetonitrile/water (9:1, v/v) solvent mixture. Thesefeatures have been exploited in a molecularly imprinted solid-phase extraction (MISPE) format, whereinthe preferential retention of stigmasterol (with an imprint factor of 12) was demonstrated with 99%recovery in comparison to cholesterol (imprint factor of 6) and ergosterol (imprint factor of 4) while inthe presence of several closely related steryl analogues.