The invention discloses a polyethylene glycol modified human thymosin beta 4 tandem repeat protein (PEG-rTbeta4), as well as a preparation method and application thereof. The PEG-rTbeta4 is obtained after recombinant human thymosin beta 4 tandem repeat protein is subjected to specific chemical modification with polyethylene glycol, wherein the rTbeta4 has gene sequence codes connected in the following sequence: a HKCDI gene sequence, a thymosin beta4 gene sequence, a GS Linker GSGSG-thymosin beta4 gene sequence and a 6*His label gene sequence. Furthermore, the invention discloses a method forpreparing the protein. The preparation method can be used for preparing target protein having the purity of 90 percent or more, has the advantages of low cost, high bioactivity and the like, and is simple and quick. Experiments prove that the PEG-rTbeta4 protein has the effects of promoting proliferation and migration of myocardial cells, resisting apoptosis, promoting hair growth and angiogenesisand accelerating wound healing. Therefore, the PEG-rTbeta4 protein has a wide application prospect in heart function recovery after myocardial infarction, hair growth promotion and wound healing acceleration.