Abstract: To elucidate the structure and function of the bim gene in Megalobrama amblycephala, this study obtained the full-length coding sequence (CDS) of bim through TA cloning and systematically analyzed the primary, secondary, and tertiary structural characteristics of Bim protein by using bioinformatics tools. Multiple sequence alignment revealed that the Bim protein of Megalobrama amblycephala exhibited the highest homology (98.34%) with that of Chanodichthys erythropterus, and a Neighbor-Joining phylogenetic tree further supported their closest evolutionary relationship. Tissue expression profiling demonstrated that both the bim gene and its putative regulatory factor mam-miR-24-3 were predominantly expressed in gill tissues compared to other organs. After 24h of hypoxia stress, their expression levels in gills were significantly upregulated, suggesting potential synergistic roles in hypoxia adaptation. Dual-luciferase reporter assays confirmed that mam-miR-24-3 directly bound to the 3′-untranslated region (3′-UTR) of bim mRNA, leading to reduced luciferase activity. When combined with fluorescence co-localization analysis using green fluorescent protein, these results indicated that mam-miR-24-3 negatively regulated bim expression at the post-transcriptional level. Subcellular localization experiments revealed that Bim protein predominantly localized to the nucleus under both normoxic and hypoxic conditions, implying its potential involvement in nuclear signaling pathways to directly regulate downstream gene transcription. This study provides a theoretical foundation for elucidating the molecular mechanisms by which bim contributes to hypoxia adaptation in Megalobrama amblycephala and lays the groundwork for screening molecular targets in hypoxia-tolerant breeding strategies.