A manufacturing method of a nitride crystalline film includes following steps. First, a substrate is provided. Next, a first nitride crystalline film is formed on the substrate. A patterned mask is then formed on the first nitride crystalline film. The patterned mask covers a first part of the first nitride crystalline film and exposes a second part of the first nitride crystalline film. Afterwards, the second part is etched, and the first part is maintained. After that, the patterned mask is removed. The first part is then etched to form a plurality of nitride crystal nuclei. Next, a second nitride crystalline film is formed on the substrate, and the second nitride crystalline film is made to cover the nitride crystal nuclei. A nitride film and a substrate structure are also provided.