The 14-3-3 family genes, highly conserved regulatory factors in eukaryotes, play critical roles in cellular regulation and stress adaptation. Despite extensive research in vertebrates and some aquatic species, the functional roles of 14-3-3 genes in bivalves remain underexplored. In this study, we identified and characterized five 14-3-3 genes (Cn14-3-3a to Cn14-3-3e) in the noble scallop Chlamys nobilis. Phylogenetic and motif analyses confirmed their high conservation, consistent with their roles in essential biological processes. Tissue-specific expression profiling revealed that these genes are predominantly expressed in immune-related tissues such as the blood, gills, and intestine, suggesting their involvement in immune responses. Furthermore, their expression dynamics under acute and chronic low-temperature stress highlighted distinct regulatory patterns: acute cold exposure from 25 °C to 9 °C elicited significant upregulation of all Cn14-3-3 genes (P < 0.05), while under chronic cold stress conditions, the expression profiles of Cn14-3-3 family members exhibited distinct patterns. Specifically, the expression levels of Cn14-3-3a and Cn14-3-3b initially showed a slight upregulation at 17 °C. However, as the temperature continued to decline, their expression gradually diminished. In contrast, the expression of Cn14-3-3c displayed a continuous downward trend with decreasing temperature. Notably, the expression of Cn14-3-3d and Cn14-3-3e remained relatively stable, showing no significant changes in response to temperature decreased. These findings provide novel insights into the functional roles of 14-3-3 genes in the cold adaptation and immune responses of bivalves, offering a foundation for strategies to enhance cold tolerance in aquaculture systems.