The novel fluorescent nanofiber films were synthesized by aminating available electrospun polyacrylonitrile nanofibers with ethylenediamine and then immobilizing fluorescein covalently through a Mannich reaction. The resulting fluorescent nanofibers have been confirmed by fluorescence spectra, field-emission scanning electron microscopy(FE-SEM) and Fourier transform infrared(FTIR) spectrum. Results show that the synthesized nanofibers exhibit strong fluorescence, which show fluorescein was immobilized covalently into the electrospun nanofiber films. Further modification of electrospun films with fluorescent molecules presents the possibility of practical sensing applications of the electrospun films.