Phage antibody library yields antibodies with higher affinity against different antigens, if diverse IgV gene repertoires can be amplified. As the currently available Fab primer sets cannot guarantee efficient amplification with high diversity, and because rare cloning sites can be found in certain Ig genes, here, we present an optimal set of Ig 5' primers, compatible with Fd 5' clone site replaced pComb3 vector, for diverse Fab phage display library construction. These novel Fab primes designed based on the newly classified IgV families, not only have best match and highest coverage for IgV family with minimized N-terminal amino acid changes, but also present good amplification diversity and efficiency of Ig gene from mice immunized with different forms of antigens (HAb18GEF, KLH-DOTA-Y, and HAb18G/pcDNA3). A high quality immune phage library with good diversity was constructed based on the mixed Ig repertoire, and five high affinity Fab antibodies were selected to specifically bind to HAb18GEF, DOTA-Y and an irrelevant antigen gamma-sm, respectively. This novel Fab primers set can be applied to the construction of diverse phage antibody library and the anti-HAb18GEF and anti-DOTA-Y Fab antibodies lay a solid foundation for radioimmunotherapy of hepatoma.