Abstract: A phage display random fifteen-peptide library expressed in the filamentous phage tail fiber protein P3N terminal was constructed using pCANTAB 5E vector. First, a random oligonucleotides fragment containing fifteen codons was designed and synthesized. The fragment was amplified by PCR. Then, the amplified target gene was digested by two restriction endonuclease of Sfi I and Not I, and connected with pCANTAB 5E phagemid vector. Finally, the recombinant vector was transformed into electro-competent cells of E. coli TG1. The capacity of peptide library was up to 5×10⁸. Twenty clones were randomly selected for sequencing; the nucleotide sequence and deduced amino acid sequences were randomized. The random fifteen-peptide library was successfully established for screening requirements.