In this paper, the influences of the growth time of low-temperature (LT) GaN nucleation layer on the crystal quality and optical properties of GaN film are investigated. It is found that the optimal LT nucleation layer growth time can effectively reduce the crystal defects and is favorable to forming the annihilation of dislocations. GaN films are grown on c-plane sapphire substrates by metal-organic chemical vapor deposition. Crystal quality and optical properties are characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution X-ray diffraction (HRXRD), and photoluminescence spectra, respectively. In the AFM images, the island density decreases as growth time increases, while the size of island becomes larger and the uniformity of island size deteriorates as growth time increases, leading to the phenomenon that the number of interfaces formed during the nucleation island coalescence, first decrease and then increase as detected by SEM, which also induces the screw dislocation density and edge dislocation density to first decrease and then increase as measured by HRXRD. This first-decrease-and-then-increase variation trend is consistent with the first-increase-and-then-decrease variation trend of the ratio of the band edge emission peak intensity to the yellow luminescence peak intensity tested by photoluminescence, which is confirmed by HRXRD. It is shown that GaN islands with different sizes and densities could lead to different mechanisms of dislocation evolution, thereby forming GaN epitaxial layers with different dislocation densities and optical properties. Through controlling the nucleation time, GaN films with the smallest dislocation density could be obtained.