Human thymosin beta 4 (TB4) is a small acidic peptide involved in angiogenesis, wound healing, cancer metastasis and cardiac repair. Currently human TB4 is synthesized chemically for research and this is costly. In order to obtain sufficient biologically active human TB4 economically, we cloned and overexpressed this protein in an Escherichia coli system. We also developed a one-step affinity purification method to purify this fusion protein. After the fusion tag was removed from the fusion protein through autohydrolysis by dithiothreitol (DTT), the biological activity and function of this recombinant human TB4 was evaluated by cell proliferation assay using prepared spleen cells and wound assay using a mouse model, respectively. Our data demonstrated that human recombinant TB4 can promote lymphocyte proliferation and differentiation. Further, it can also promote full-thickness cutaneous wound healing in BALB/c mice. To our knowledge, this is the first report of recombinant human TB4 with the ability to promote wound healing.