Molecular crowding is a new approach to promoting molecular imprinting more efficiently. In this work, this concept was applied to the preparation of imprinted monoliths in the presence of molecular crowding agent for stabilizing binding sites and improving molecular recognition. The imprinted monolithic column was synthesized using a mixture of enrofloxacin (template), methacrylic acid, ethylene glycol dimethacrylate, and polystyrene (molecular crowding agent). Some polymerization factors, such as template-monomer molar ratio and the composition of the porogen, on the imprinting effect of resulting MIP monolith were systematically investigated. Moreover, a stoichiometric displacement model for retention was successfully applied to evaluate the interaction between the solute and the stationary phase. Compared with the MIP prepared by conventional polymerization, the molecular crowding-based monolithic MIPs showed higher selectivity. The results suggested that molecular crowding is a powerful strategy to increase the effect of molecular imprinting.