The widespread use of chlordecone (CLD), an organochlorine pesticide, until the 1990s to protect banana crops in the French West Indies led to significant pollution of water and soil and, subsequently, of bovine intended for human consumption. Carcasses are submitted to official controls based on perirenal fat CLD determination. In order to allow for pre-slaughter controls, a selective analytical method based on a molecularly imprinted polymer (MIP) associated to the LC/MS-MS method was developed to determine the level of CLD in bovine serum that can be collected before slaughter. Different synthesis conditions were therefore assayed by varying the nature of the monomer and of the porogen, and the most promising MIP in terms of selective retention for CLD (extraction recovery close to 100%) was completely characterized by solid-phase extraction (repeatability of the extraction procedure, of the synthesis, and of the cartridge filling) in pure medium. The capacity of the MIP was determined at 0.13 µmol g−1 of MIP. After application of a spiked bovine serum sample on the MIP, the selective retention was maintained (87 and 21%, respectively, on the MIP and on the corresponding non-imprinted polymer). Moreover, extraction on the MIP led to a cleaner extract compared to those issued from a conventional C18 sorbent.