Objective To predict the structure and function of the protein encoded by Mycobacterium tuberculosis gene Rv3529c through bioinformatics software and perform prokaryotic expression of the protein. Methods The secondary and tertiary structure, physicochemical properties, hydrophobicity, signal peptide, transmembrane domain, subcellular localization and immune epitopes of Rv3529c-encoded protein were analyzed and predicted through bioinformatics prediction software such as The UniproKB database, PredictProtein service, AlphaFold system, SWISS-MODEL service, ProtParam protein analysis tool, ProtScale, DeepTMHMM service, SignalP-5.0 service, ProtCompB method, IEDB database and STRING database. Expression plasmid vector pET-32a-Rv3529c was constructed and performed prokaryotic expression by molecular cloning technique. Amino acid sequences were compared by the Blast tool of UniprotKB database, and evolutionary tree was constructed by MEGA 11 software for phylogenetic analysis. Interactions among proteins were analyzed with STRING database. Results Gene Rv3529c was 1 155bp in length and encoded 384 amino acids. Gene Rv3529c encoded a protein with a molecular weight of 43.35 kDa and an isoelectric point of 6.04, and its secondary structure was mainly α-helixi, which was a stable hydrophilic protein without transmembrane domain and signal peptide. It was predicted that its subcellular location was in cytoplasm or cell membrane, with multiple antigen-antibody epitopes, and interacted with various proteins. The protein Rv3529c with high concentration and purity was obtained after cloning, expression, identification and purification. Conclusion The protein Rv3529c of Mycobacterium tuberculosis is successfully cloned, expressed and purified, and its structure and function is tentatively predicted.