A method was presented, which allowed for Zero-valent copper (Cu-0) nanoparticles fabricated in/on polyacrylonitrile (PAN) nanofibers by two-step including the preparation of the copper(II)/polyacrylonitrile (Cu2+/PAN) nanofibers by electrospinning, and the preparation of the Cu-0/PAN nanofibers by high-pressure hydrogenation reduction. The Cu-0/PAN nanofibers were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), Fourier transform infrared (FT-IR), and X-ray photoelectron spectroscopy (XPS). The results showed that the copper nanoparticles were successfully synthesized in the composite fibers. Furthermore, antibacterial tests indicated that the Cu-0/PAN nanofibers have good bactericidal effects.