Abstract: In order to study the enzymatic characteristics and structure of prolyl endopeptidase from Haliotis discus hannai (Hdh-PEP), recombinant PEP was cloned and highly expressed in E.coli. Hdh-PEP with molecular weight of 85 kDa was successfully purified and its surface hydrophobicity was greatly affected by pH at low value (pH2～6) and temperature (20～60 ℃). Amino acid sequence homology analysis showed that there were three highly conserved sequences in the catalytic domain of Hdh-PEP: Seq 1: K-D-G-T-K/R-I-P, Seq 2: Y-G-Y-G-G-F and Seq 3: I-R-G-G-E-Y/F. Kinetic study revealed that the K_m and k_cat of Hdh-PEP were 5.32 μmol/L and 15.7 s⁻¹, respectively. Specific inhibitors SUAM-14746 and ZPP of PEP had strong inhibition on Hdh-PEP activity, and serine protease inhibitor (PMSF) also exhibited inhibition. A high specific polyclonal antibody toward Hdh-PEP was prepared which could be applied for the detection of native PEP in abalone muscle. The successful in vitro expression of Hdh-PEP and preparation of a specific polyclonal antibody against Hdh-PEP provided an important reference for subsequent investigation on Hdh-PEP.