A simple method for the formation of molecular recognition sites in polymer nanofibers was developed. Electrospun nanofibers were prepared from a Solution mixture of poly(ethylene terephthalate) (PET) and polyallylamine in the presence of a template molecule, 2,4-dichlorophenoxyacetic acid (2,4-D). The polyamine was used to provide functional groups that interact with the template during the electrospinning process, and PET acted as a major supporting matrix to ensure easy fiber formation and to minimize the conformational change of the polymers when the nanofibers were subjected to different solvent treatments. When the template was removed by solvent extraction, imprinted binding sites were left in the nanofiber materials that are capable of selectively rebinding the target molecule. The molecularly imprinted nanofibers were characterized by SEM, FT-IR, and radioligand binding analysis.