Thymosin-α1 (Tα1), as a commercial peptide drug, has been chemically synthesized and widely used for enhancing immune responses and anti-tumor. The increasing therapeutic Tα1 need leads to concerns about its mass and low-cost production processes. To prepare peptide thymosin-α1 by a novel recombination expression and purification technique. The thymosin-α1 was designed into a tetraploid concatemer by genetic manipulation and expressed in E. coli, and the purified concatemer thymosin α1 was obtained by one-step heating. Subsequently, the concatemer thymosin-α1 was cleaved into monomers with cyanogen bromide dissolved in 50%-70% trifluoroacetic acid. The thymosin-α1 monomer was further purified by high performance liquid chromatography with purity ≥ 98%. Finally, the obtained thymosin-α1 could stimulate lymphocyte proliferation, which was almost as effective as the commercial thymosin-α1 (Zadaxin?).Finally, The recombinant thymosin-α1 was successfully obtained, which is similar to the commercial one, by genetic recombination, heat purification and appropriate cleavage.