Fluorescein/polyvinyl pyrrolidone (PVP) composite nanofibers with different fluorescein loadings (with a weight concentration of 0-5.0%) are fabricated via electrospinning. Morphologies, structures and photoluminescent (PL) properties of these straight, helical or wavelike fibers are characterized by scanning electron microscopy (SEM), fluorescence microscopy and a spectrophotometer. It is found that the maximum emission of the as-spun fluorescein/PVP fibers occurs at 510 nm. The PL intensity of the composite fiber increases with fluorescein concentration, then fluorescence quenching appears when the concentration reaches 1.67%. The mechanism of fluorescence quenching of fluorescein is discussed. In addition, the composite fibers exhibit a much stronger PL intensity than fluorescein/PVP bulk film owing to larger specific surface area, which makes them promising materials for biomedical applications such as probes and sensors.