Abstract: 【Objective】The purpose of the study was to conduct the protein sequence analysis and expression characteristics studies of Defensin-2（Def-2） protein of Apis mellifera, in order to provide a theoretical references for exploring the immune defense mechanism, development and metabolic pathway of defensin gene in A. melliferia. 【Method】Bioinformatics analysis websites and software were used to predict and analyze Def-2 protein, while the gene relative expression levels of A. mellifera at different developmental stages（larva stage,pupa stage and 1,5,10,15,20,25 and 30 d adult stage worker bee） were analyzed by real-time fluorescence quantitative PCR. 【Result】The Def-2 gene of A. mellifera encoded 104 amino acids residues with a relative molecular weight of 11858.90 Da and an isoelectric point（pI）of 8.54, identifying it as an amphipathic, unstable protein. This protein had a signal peptide of 21 amino acids at its Nterminus and had one transmembrane domain. The subcellular location was primarily in the endoplasmic reticulum, vacuoles, and Golgi body. There were no glycosylation sites and only 15 phosphorylation sites. The secondary structure of this protein was dominated by random coil structure（54.81%）, with large distribution of extended chain（28.85%）and a few α-helix（16.35%）. The protein sequence was highly conserved and interacted genetically with proteins such as Def-1, GB44032-PA, Dl-2 and Imd, and contained defense protein-like domain（DEFL）,which adopted a structure characterized by a cysteine-stabilized α/β scaffold. The phylogenetic relationship showed close similarity to A. florea. The expression level of Def-2 gene of A. mellifera varied at different developmental stages and increased with age, the relative expression of 30 d was significantly higher than the other days（P＜0.05）. 【Conclusion】Def-2 protein of A. mellifera is an amphipathic unstable protein,which is involved in intercellular signal transmission and maintains normal cell morphology. The protein sequence is highly conserved, with the closest phylogenetic relationship to A. florea. The gene is expressed in high abundance at 30 d of adult A. mellifera, suggesting that it may regulate immune gene transport and affect the immune recognition process of A. mellifera.