We present a systematical study on the evolution of antiphase boundary (APB) densities in Fe3O4 films, which were prepared by pulsed laser deposition and post annealing at different temperatures. By measuring the electron-phonon coupling parameter and using Allen's formula, we evaluate the films' antiphase boundary densities, which show a decreasing tendency with increasing annealing temperature. Consequently, a 50% increase of spin polarization in Fe3O4 films is achieved, and a 110% increase of the magnetoresistance ratio was found in 900 °C annealed Fe3O4 films compared to the as-grown sample. This work could contribute to the effective manipulation of APB densities and spin polarization in Fe3O4 films, which is desirable for the application of spintronics devices based on Fe3O4 films.