A versatile method for preparing a reverse phase/weak anion exchange/hydrophilic interaction trimodal polymer monolith was proposed by the one-step copolymerization of amino acid-based monomer and N,N′-methylenebisacrylamide. The retention mechanisms of the resultant monolith were evaluated using different series of small molecules. Furthermore, the separation of protein mixtures could be easily achieved under isocratic elution. The proposed polymer monolith, which has the merits of easy preparation, low mass transfer resistance and good biocompatibility, is a promising separation medium for small and large molecules.