Accurate determination of the atomic spatial configuration of Frenkel defects is important for understanding the mechanism and fully utilizing these defects to optimize the material properties. In this study, aberration-corrected scanning transmission electron microscopy (STEM) was used to identify the Fe vacancies and Fe Frenkel defect pairs, which have not been previously investigated, in NiFe2O4 (NFO). The spatial distribution of these point defects is determined by comparing the experimental and simulated images, where the experimental image intensities are consistent with the calculated image intensities. We confirmed the stabilities of the observed point defect configurations and calculated their electronic structures using density functional theory. A comprehensive understanding of the relationship between the Frenkel defect spatial configurations and electronic properties is obtained, which provides an alternative method to regulate the NFO performance.