In this study, we aimed to examine the relationship between the pancreatic alpha-amylase gene 5' flanking sequence and the feeding habits of fish by analyzing the differences of alpha-amylase gene transcription factors in different fishes. Pancreatic alpha-amylase gene 5' flanking sequences were obtained from a total of 32 fish species. Five of them were obtained by PCR and the remaining were obtained by searching the NCBI database. The length of the obtained 5' flanking sequences was 824 bp. Then, the transcription factors and phylogenetic relationship analyses were carried out for these pancreatic alpha-amylase gene 5' flanking sequences. Fishes were divided into herbivores, omnivores, and carnivores, according to their different trophic positions. The percentage similarity analysis was used to study the differences in the composition of transcription factors in fishes with different feeding habits. The similarity of the pancreatic alpha-amylase gene 5' flanking sequence among different fishes was very low. The pancreatic alpha-amylase gene 5' flanking sequences of 32 fish species contained a species transcription factor that ranged from 40 to 98. The results of the percentage similarity analysis showed that there were differences in the transcription factors of pancreatic alpha-amylase gene 5' flanking sequences among fishes with different feeding habits. Differences between the Herbivore and Carnivore groups were mainly found in the E47, C/EBPalpha, NF-Y, and Pax-2 transcription factors. Differences between the Herbivore and Omnivore groups were mainly found in the deltaEF1, MyoD, NF-Y, AREB6, and Pax-2 transcription factors. Differences between the Omnivore and Carnivore groups were mainly found in the GATA-1, SRY, MyoD, HFH-8, AREB6, Pax-2, STAT5A, and AP-1 transcription factors. These transcription factors play an important role in glucose metabolism and can enhance or inhibit gene expression. In liver glucose and lipid metabolism, the E47 and GR transcription factors act jointly as metabolism promoters and enhancers. In the regulation of gluconeogenesis, C/EBPalpha and FOXO1 act together on the IRE controlled promoter to regulate the expression of target genes such as PCK1 and G6PC. Transcription factor NF-Y plays an important role in promoting glucose metabolism. The phylogenetic tree results were generally consistent with the traditional morphological taxonomy. Fishes with the same feeding habits did not cluster together in the phylogenetic tree. The results indicated that there were several transcription factors in the pancreatic alpha-amylase gene 5' flanking sequences that were related to the feeding habits of fish. There is a certain relationship between transcription factors and the dietary differentiation of fish.