We report the layer-by-layer (LbL) surface functionalization of high aspect ratio (>100000:1) polystyrene (PS) electrospun fibers (ESFs) of various diameters (400 nm or 1.5 μm) with polyelectrolytes (PEs), deoxyribonucleic acid (DNA) oligonucleotides, and PE/gold nanoparticle (AuNP) composite layers. ESFs were coated with poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) multilayers, the thickness of which was controlled by adjusting the number of PE layers deposited. PAH/PSS multilayer buildup was demonstrated by dissolving the inner PS fibers with tetrahydrofuran, resulting in the formation of hollow PAH/PSS fibers. DNA multilayer coated ESFs were also prepared using oligonucleotides with repeating blocks of adenosine and guanosine (polyA15G15) and thymidine and cytidine (polyT15C15). The polyT15C15 was labeled with tetramethylrhodamine. Linear DNA multilayer buildup on the ESFs was observed through an increase in fluorescence intensity using confocal laser scanning microscopy. Final...