PUBLISHED Reflectance anisotropy spectroscopy (RAS) has been used to measure the optical anisotropies of bulk and thin-film Fe3O4(110) surfaces. The spectra indicate that small shifts in energy of the optical transitions, associated with anisotropic strain or electric field gradients caused by the (110) surface termination or a native oxide layer, are responsible for the strong signal observed. The RAS response was then measured as a function of temperature. A distinct change in the RAS line-shape amplitude was observed in the spectral range from 0.8 to 1.6 eV for temperatures below the Verwey transition of the crystal. Finally, thin-film magnetite was grown by molecular beam epitaxy on MgO(110) substrates. Changes in the RAS spectra were found for different film thickness, suggesting that RAS can be used to monitor the growth of magnetite (110) films in situ. The thickness dependence of the RAS is discussed in terms of various models for the origin of the RAS signal.