Antiphase boundaries in spinel ferrites have been considered as the functional elements with great potential for applications in spintronic devices. Engineering the antiphase boundaries is a big challenge in experiments. Here, we report on that a high density of antiphase boundaries in CuFe2O4 films have been prepared on rough surface of MgAl2O4 (001) substrates. By means of advanced electron microscopy these antiphase boundaries are found to be of {011}1/4 < 011 >-type and bound by interfacial dislocations with Burgers vector of a/4 < 011 > that contribute to relaxation of in-plane and out-of-plane lattice strain in the heterostructure. Our results provide evidence that the rough substrate surface can be applied for the formation of antiphase boundaries in the CuFe2O4/MgAl2O4 system to tune the magnetic and electrical transport properties of epitaxial spinel ferrite films.