Three-dimensional spherical assemblies of Ni-doped ZnO nanocrystals have been prepared by the solution phase synthesis process. It has been observed that transition metal ions (Zn, Ni) are uniformly distributed in the sample and exist in the +2 oxidation state. Detailed investigation of structural defects formed during the formation of spherical assemblies by oriented attachment of nanocrystals was carried out by high-resolution transmission electron microscope (HRTEM), Raman and photoluminescence (PL) spectroscopy. HRTEM analysis revealed the existence of various crystal defects, such as stacking faults, dislocations, etc. The incorporation of Ni2+ into ZnO structure strongly influences the vibrational and optical properties of the sample due to the increment of defect densities. Compared to the optical phonons of ZnO, additional mode observed at 538 cm−1 in Raman spectra of Ni-doped ZnO could be associated with the incorporation of Ni2+ in Zn2+ site. The increase in PL intensity of green emission with Ni2+ doping indicates the formation of a higher concentration of oxygen vacancy in doped nanostructures.