This study aimed to investigate the relationship between trypsin and the feeding habits of live prey fish in Siniperca chuatsi and to elucidate the transcriptional regulatory mechanism of the key trypsin gene in S. chuatsi. In this study, we used bioinformatics methods to conduct quantitative, structural, and phylogenetic analyses of trypsin genes in 11 fish species with different feeding habits, combining qPCR, dual-luciferase reporter, and trypsin activity assays to analyze the transcriptional regulation mechanism of the key trypsin gene subtype in S. chuatsi and its correlation with trypsin activity at the first feeding stage. The results showed that a total of 13 trypsin genes distributed at six loci on four chromosomes were identified in S. chuatsi, which was more than that in omnivorous and herbivorous fish, indicating that the amplification of trypsin genes in carnivorous fish is highly adapted to the food environment. Carnivorous S. chuatsi was amplified at key gene loci 2 (SC7-LG17-21898 and SC7-LG17-21899) and 3 (SC7-LG17-21428, SC7-LG17-21429-1, SC7-LG17-21429-2, SC7-LG17-21430-1, and SC7-LG17-21430-2), which are homologous to the key trypsin gene PRSS1 in humans. More significant gene amplification occurred at locus 3, where SC7-LG17-21430-2 had a significantly higher expression level than the other subtypes, indicating that it was the key trypsin subtype in S. chuatsi. The dual-luciferase reporter assay results showed that the promoter activity of three constructed deleted fragments in the 5' flanking region of the SC7-LG17-21430-2 subtype had significant differences, and the region of –593 bp−+20 bp had the highest promoter activity, suggesting it as the core promoter region. Furthermore, PDX1, a transcription factor associated with early pancreatic development, was barely expressed in S. chuatsi at 3 dph and was observed in the core promoter region. Moreover, point mutations in the binding site of PDX1 result in a significant decrease in the promoter activity of the core region. The trypsin activity of S. chuatsi at 3 dph was significantly lower than that of Danio rerio, indicating that the low expression of pdx1 might lead to the low expression of SC7-LG17-21430-2 and low trypsin activity, which could be associated with the unique feeding habits of live prey fish in S. chuatsi. These results provide the groundwork for analyzing the mechanisms underlying the special feeding habits of S. chuatsi.