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<p>Thymosin alpha 1 (T&#945;1) has immunomodulatory and anti-tumor effects in patients and has been commercialized in worldwide. An innovative technique is therefore impending to achieve high-yield expression and purification of T&#945;1 to meet the increasing requirements for clinical applications. T&#945;1 can enhance T cells, dendritic cells and antibody responses, and also augment an anti-tumor immune response. In the current study, we developed a novel technique to produce T&#945;1 concatemer and investigated its capability in anti-tumor immunotherapy. We expressed the recombinant 2&#215;T&#945;1 concatemer protein (T&#945;1&#9313; protein) in <i>Escherichia coli. </i>The purity of T<i>&#945;</i>1&#9313; was higher than 95% as assessed by HPLC analysis. In vitro, T&#945;1&#9313; could stimulate the proliferation of mouse splenic lymphocyte, and increase the apoptosis of tumor cell lines. In vivo, T&#945;1&#9313; significantly inhibited the tumor growth in B16 tumor-bearing mice. Compared with T&#945;1, the T&#945;1&#9313; is of more effective bioactivity than T&#945;1. The purified T<i>&#945;</i>1&#9313; is a promising substitute for synthetic T<i>&#945;</i>1 because of its potent anti-tumor effects. We concluded that the expression system for T<i>&#945;</i>1 concatemer was constructed successfully, which could serves as a highly efficient tool for the production of large quantities of the highly active protein.</p>