Objective To isolate key sequences from a phage display 12-mer peptide library, synthesize transforming growth factor-beta1 (TGF-β1) bioactive peptides and evaluate the fibroblasts affinity of TGF-β1 bioactive peptides. Methods Using monoclonal anti-human TGF-β1 antibody as the target, a phage display 12-mer peptide library was screened and target sequences were isolated. The key sequences of TGF-β1 were chosen. The key sequences were then modified to generate TGF-β1 bioactive peptides. Immunofluorescence assay was performed to evaluate the fibroblasts affinity of TGF-β1 bioactive peptides. Results Ten peptides similar to TGF-β1 were isolated from a phage display 12-mer peptide library. Seven key sequences of TGF-β1 were chosen and then modified to generate TGF-β1 bioactive peptides. TGF-β1 bioactive peptides were then purified to reach a purity of 98%. The C terminal of TGF-β1 bioactive peptides was closed, and the N terminal was labeled with rhodamine dye laser. Immunofluorescence assay showed one TGF-β1 bioactive peptides was able to combine with the fibroblasts. Conclusion The key sequences of TGF-β1 can be screened from a phage display 12-mer peptide library. One TGF-β1 bioactive peptides can combine with the fibroblasts. The results are expected to help promoting wound healing.