In this study, we used electrospinning to fabricate nucleobase-functionalized and photo-cross-linkable poly[1-(4-vinylbenzyl uracil)] (PVBU) nanofibers. PVBU of high-molecular-weight (M-n > 250 550 g/mol) possessed a high thermal stability and sufficient chain entanglement to produce uniform fibers without forming beads. These uracil-functionalized nanofibers were further photo-cross-linked through exposure to UV light at a wavelength of 254 nm. After immersing in N,N-dimethylacetamide, the pristine PVBU fibers dissolved, while the cross-linked PVBU fibers maintained their shape; thus, the cross-linked PVBU nanofibers exhibited good dimensional stability and improved solvent resistance.