Neuropeptide Y (Npy) is an intricate neuropeptide regulating numerous physiological processes. It is a highly conserved peptide known to improve feed intake in many vertebrates, including fishes. To enlighten the mechanism of Npy in spotted scat feed intake control, we cloned and identified the Npy cDNA sequence. We further examined its expression in some tissues and explored its expression effects at different time frames (hours and days). Here, we discovered that spotted scat Npy comprised a 300 bp open reading frame (ORF) and a 99 amino acid sequence. Npy was identified to be expressed in all tissues examined. Using in situ hybridization examination, we proved that npy has a wide expression in the brain of the spotted scat. Furthermore, the expression of npy in the hypothalamus significantly increased one hour after feeding (p < 0.05). Further, it was revealed that npy expression significantly increased in fish that were fasted for up to 5 days and significantly increased after refeeding from the 8th to the 10th day. This suggests that Npy is an orexigenic peptide, and hence, it increases food intake and growth in the spotted scat. Additionally, results from in vitro and in vivo experiments revealed that Npy locally interacts with other appetite-regulating peptides in the spotted scat hypothalamus. This research aimed to set a fundamental study in developing the feed intake regulation, improving growth and reproduction, which is significant to the aquaculture industry of the spotted scat.