Single-handed helical phenolic resin nanofibers were synthesized through a supramolecular templating approach using the supramolecular self-assemblies of a pair of chiral low-molecular-weight amphiphiles as the templates and 2,4-dihydroxybenzoic acid and formaldehyde as the precursors. The phenolic resin nanofibers were characterized using field-emission scanning electron microscopy, transmission electron microscopy and diffused reflection circular dichroism. The results indicated that the chirality of the supramolecular self-assemblies was successfully transferred to the phenolic resin nanofibers. The left- and right-handed helical phenolic resin nanofibers exhibited opposite optical activity.