In this work, a molecularly imprinted monolithic column was synthesized by a facile procedure and was applied for specific recognition of domoic acid, an amnesic shellfish poison. The poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) molecularly imprinted monolith was synthesized in a stainless steel column by in situ polymerization. Pentane-1,3,5-tricarboxylic acid was used as a dummy imprinting template instead of the highly toxic and expensive target molecule. It is the first time that a molecularly imprinted monolith is introduced for separation and detection of domoic acid. After optimizing the preparation conditions, the prepared imprinted monolith was systematically characterized and exhibited excellent stability and permeability as a HPLC stationary phase. The results of chromatographic analysis demonstrated that the molecularly imprinted monolith exhibited specific retention and selective recognition toward domoic acid, with an imprinted factor up to 3.77. Furthermore, the molecularly imprinted monolith was successfully applied for selective enrichment of domoic acid from biological samples. Graphical abstract A molecularly imprinted monolith was prepared by using a dummy imprinted template and was successfully applied for specific recognition of domoic acid.