Abstract: In order to construct a multi-epitope candidate antigen based on Mycobacterium bovis Ag85B protein, in the experiment, bioinformatics methods were used to analyze the physicochemical properties, hydrophilicity/hydrophobicity, and secondary structure of Ag85B protein, and to predict its antigenic epitopes for T and B lymphocytes. A multi-epitope candidate antigen was constructed into the vector pET-28a（+）, and the physicochemical properties, antigenicity and immunosimulation of the multi-epitope candidate antigen were analyzed and predicted. The results showed that Ag85B protein had 325 amino acid residues, relative molecular weight of 34.580 85, theoretical isoelectric point of 5.62, and fat coefficient of 72.18; it subcellularly localized in mitochondria, cytoplasm and Golgi, with hydrophilic, signal peptide and transmembrane structural domains; it belonged to the secretory protein; the percentages of irregularly curled and β-turned structures were 28.15% and 8.62%, respectively, and it contained three T-lymphocyte dominant epitopes and eight B-lymphocyte dominant epitopes. The constructed multi-epitope candidate antigen of Ag85B was hydrophilic protein and non-allergen; the two predicted immunogenicity parameters were 0.893 and 1.524, respectively; it could induce specific humoral and cellular immune responses. The results indicated that the test successfully constructed a multi-epitope candidate antigen based on Mycobacterium bovis Ag85B protein, and the multi-epitope candidate antigen might induce T and B lymphocyte immune responses in the organism.