Changing the film thickness to manipulate the microstructural properties has been considered as a potential method in practical application. Here, we report that atomic-scale structural properties regulated by film thickness in NiCO2O4(NCO)/CuFe2O4(CFO) bilayer heterostructure prepared on (001)-MgAl2O4 (MAO) substrate by means of aberration-corrected scanning transmission electron microscopy (STEM). The misfit dislocations at NCO/CFO interface and antiphase boundaries (APBs) bound to dislocations within the films both are found in NCO (40 nm)/CFO (40 nm)/MAO heterostructure to contribute to relaxation of mismatch lattice strain. In addition, the non-overlapping a/4[101]-APB is found and the structural transformation of this kind of APB is resolved at atomic scale. In contrast, only the interfacial dislocations form at interface without the formation of APBs within the films in NCO (10 nm)/CFO (40 nm)/MAO heterostructure. Our results provide evidence that formation of microstructural defects can be regulated by changing film thickness to tune magnetic properties of epitaxial bilayer spinel oxide films.