Photoluminescent fullerene nanoparticles/ nanofibers have potential applications in bioimaging. A novel fluorescent nanofibrous material, consisting of fullerene nanoparticles and poly(L-lactide) (PLLA), was fabricated via a simple electrospinning method, and the composite nanofibers were characterized by various techniques such as scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM), and transmission electron microscopy (TEM). The nanofibers were uniform, and their surfaces were reasonably smooth, with the average diameters of fibers ranging from 300 to 600 nm. The fullerene nanoparticles were encapsulated within the composite nanofibers, forming a core shell structure. The nanofiber scaffolds showed excellent hydrophilic surface due to the addition of water-soluble fullerene nanoparticles. The composite nanofibers used as substrates for bioimaging in vitro were evaluated with human liver carcinoma HepG2 cells, the fullerene nanoparticles signal almost displayed in every cell, implying the potential of fluorescent fullerene nanoparticles/PLLA nanofibers to be used as scaffolds for bioimaging application.