In this paper, NiFe2O4 powders were prepared by a thermal decomposition process with various temperatures. The structural characteristic and intrinsic defect were characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet–visible (UV–vis) spectroscopy, and photoluminescence (PL). The morphology was investigated by scanning electron microscope (SEM) and transmission electron microscope (TEM). XRD pattern reveals the pure NiFe2O4 phase as the calcination temperature of 900 and 1,000 °C. The calcination of 900 °C depicted the micro-octahedral structures with a smooth surface. The calculated hopping length and the FT-IR spectra reveal the cation shifting from octahedral to tetrahedral structure. The decreasing energy bandgap of NiFe2O4 was investigated as the calcination temperature increased. The PL transition of 3A2g(3F) → 3T1g(3F) refers to the band-to-band emission. The structural characteristics, morphology, and defects were investigated and discussed in terms of phase, bonding vibration, crystal growth mechanism, emerging energy level and electron transition.