The growth and characterization of high-quality ultrathin Fe3O4 films on semiconductor substrates is a key step for spintronic devices. A stable, single-crystalline ultrathin Fe3O4 film on GaAs(001) substrate is obtained by post-growth annealing of epitaxial Fe film with thicknesses of 5 and 12 nm in air. Raman spectroscopy shows a high ability to convincingly characterize the stoichiometry, epitaxial orientation and strain of such ultrathin Fe3O4 films. Polarized Raman spectroscopy confirms the unit cell of Fe3O4 films is rotated by 45° to match that of the Fe (001) layer on GaAs, which results in a built-in strain of − 3.5% in Fe3O4 films. The phonon strain-shift coefficient(−126 cm−1) of the A1g mode is proposed to probe strain effect and strain relaxation of thin Fe3O4 films on substrates. It can be used to identify whether the Fe layer is fully oxidized to Fe3O4 or not. Copyright © 2011 John Wiley & Sons, Ltd.