Calcineurin B-like (CBL) proteins are plant-specific calcium sensors critical for ion homeostasis and stress tolerance. Here, seven MdCBL genes were genome-wide identified in apple (Malus domestica). We conducted systematic bioinformatic profiling of their physicochemical features, subcellular localization, cis-regulatory elements, phylogeny, secondary structures, phosphorylation sites, and functional annotations and further verified the salt-stress regulatory function of MdCBL5 via transgenic tests. MdCBL proteins contain 210–246 amino acids, with molecular weights of 24,196.73–28,283.39 Da, pI values of 4.63–4.97 and instability indices of 37.12–49.20. Localization prediction placed these proteins in nuclei, cytosol and chloroplasts. Their promoter regions are rich in hormone-responsive (auxin, ABA, salicylic acid) and stress-responsive (cold, drought, salt) cis-elements. Phylogenetic clustering divided MdCBLs into five subgroups (A–E) with high homology to Arabidopsis CBLs. Random coils and α-helices dominate their secondary structures, and serine residues constitute most phosphorylation sites. Functional annotation supports their involvement in calcium signaling, ion transport and diverse stress adaptation. Salt stress experiments have confirmed that MdCBL5 may enhance apple salt resistance by promoting MdSOS gene expression. Meanwhile, transient transformation in apple fruit showed that MdCBL5 can effectively enhance fruit quality traits. Collectively, this study establishes a theoretical foundation for further elucidating the biological functions of the apple MdCBL5 gene and provides valuable insights for genetically improving stress resistance and fruit quality in apple via molecular breeding strategies.