Total 26 β-defensins were identified and characterized from Chinese soft shell turtle (Pelodiscus sinensis) EST library. The importance of Ps-BDs in turtle’s innate immune response could be deduced from the expression profiles. Ps-BDs are predicted to mainly adopt β-strands and/or α-helix conformations homology modeled by Rosetta. Ps-BD2 with the best character traits of β-defesin was expressed in the eukaryotic expression system. The recombinant Ps-BD2 showed strong antimicrobial activity, but negligible hemolytic and cytotoxic activities.