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 (polyT 15C15). 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. Finally, nanocomposite PE/gold fibers were prepared by immersing the PAH/PSS-coated PS fibers into a dispersion of 4-(dimethylamino) pyridine-stabilized AuNP. This resulted in a dense packing of Au NP in the PE film localized on the PS fiber surface. These fibers have potential application in sensing, filtration, and catalysis and as release materials in medicine and agriculture.