A series of Zn and Co-substituted nickel ferrite nanoparticles of nominal composition Ni1?xZnxFe2?xCoxO4 (x = 0, 0.1, 0.2, 0.3, 0.4, 0.5) have been synthesized by the PVA assisted sol–gel method. The reported structural and magnetic properties of the samples were investigated by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM). Also, the effect of these substitutions on vacancy distribution is investigated with positron annihilation lifetime spectroscopy (PALS). The cation distribution is inferred from the X-ray diffraction using the MAUD program to investigate ion occupancy, the exact coordinates of the atoms and unit cell dimensions. XRD analysis using the Rietveld refinement technique proves the crystalline structure and the growth of pure, single-phase and nanosize substituted nickel ferrite. These results are confirmed by the performed FESEM and FTIR studies. Magnetic measurements revealed that the values of the saturation magnetization (Ms) in the Ni1?xZnxFe2?xCoxO4 samples gradually increased for doping levels up to x = 0.4, then decreasing for further content. Also, the coercivity showed an overall decreasing trend with increasing doping. The positron lifetime spectra of the substituted nickel ferrite were decomposed into three-lifetime components, ?1, ?2, ?3. First of all, ?1, is depends on the positrons that do not get trapped by the vacancy defects. The second lifetime component, ?2, is attributed to positrons annihilation in vacancy-type defects in ferrite nanoparticles. The final lifetime, ?3, is due to the annihilation of positrons across nanoparticles in the free volume of ferrite structure. Also, magnetic behaviors could be explained by positron annihilation lifetime spectroscopy.