This paper presents the positron study on carbon-Fe3O4 coaxial nanofibers, which is one kind of new promising functional materials. According to the positron annihilation lifetime spectra and the coincidence Doppler broadening spectra, we find in this material that positrons annihilate partly in monovacancies of Fe3O4, partly in microcavities of Fe3O4 and partly in shelled carbon nanotubes. We also estimate the annihilation proportion in each part, which reveals the nanofibers’ microstructure to a certain extent.