Abstract: A strategy to construct a single chain variable fragment antibody combinatorial library was carried out through three fragment ligation using self-designed primers. Sequence information of antibody variable heavy and light fragments was used to design 2 sets of primers with the appropriate restriction endonuclease sites to facilitate the clone construction. This alternative approach allows construction of single chain variable fragment (scFv) phage display library through a simple three-fragment ligation process and allows inclusion of any desired detection marker at the same time.