This review article deals with molecularly imprinted polymers (MIPs) as affinity-based separation media for sample preparation. An over view of two types of MIPs (molecularly imprinted particle and monolith) used for the sample preparation and modes of molecularly imprinted SPE (online mode, offline mode, on-column extraction, SPME, and microextraction in packed syringe) is given, focusing on the advantages and disadvantages of these types and modes. Next, problems (template leakage and incompatibility with aqueous conditions) associated with molecularly imprinted SPE and how to overcome those problems are described. Finally, pharmaceutical, food, bioanalytical, and environmental application of molecularly imprinted SPE will be discussed.