Defensins are a group of host defense peptides that play a central role in host innate immune responses. Here, 26 genes encoding β-defensin-like peptides have been identified for the first time in Pelodiscus sinensis using database mining approach. Phylogenetic study confirmed that β-defensins are fast evolving genes with high rates of sequence substitutions. The expression level of several selected genes in different tissues was examined by RT-PCR. Ps-BDs mainly adopt β-strands and/or α-helix conformations homology modeled by Rosetta program. Further, Ps-BD2 was expressed in Pichia pastoris and purified using Ni-NTA column and RT-HPLC. As expected, the rPs-BD2 showed strong antimicrobial activity, but displayed a negligible hemolytic and cytotoxic activity on human erythrocytes and Raw 264.7 murine macrophage cells, respectively. Our results suggested that the Ps-BD2 was produced efficiently in P. pastoris expression system, which makes the large-scale use of rPs-BDs possible in the future clinical practice.