This study presented a hydrophobic interaction-based poly(HEMA-MATrp) monolithic chromatographic column (MCC) to remove amoxicillin from aqueous solutions. In addition to their porous structure, monolithic-filled columns offer superior properties without loss of performance, which is one of the points that make them unique. The specific surface area of the monolithic column synthesized by the bulk polymerization of 2-hydroxyethyl methacrylate and N-Methacryloyl-L-tryptophan. Also, poly(HEMA- MATrp) MCC has been characterized via FTIR, SEM, and elemental analysis. According to BET analysis, the specific surface area of the poly(HEMA-MATrp) monolithic chromatographic column (MCC) is 14.2 mg/g. The adsorption and desorption of amoxicillin in an aqueous solution were investigated comparatively in both continuous fixed bed and batch adsorption. The highest adsorption value of amoxicillin was determined at pH 7 in the presence of PBS as 62.11 mg/g. The appropriate adsorption isotherm for the adsorption of amoxicillin was Langmuir, and the reaction kinetics was pseudo-second-order. No significant loss was observed for the adsorption capacity of poly(HEMA-MATrp) MCC after the 5 cycles of adsorption-desorption studies. Also, the loss for the adsorption capacity of the monolithic column is just %5.2 after 6-month storage, proving the reusability and storability of the monolithic column.