Prolyl endopeptidase (PEP) is a serine protease which has been implicated in many biological processes, such as learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PEP participates in mammalian reproductive process by specifically degrading peptide hormones and gonadal hormones containing proline residues. Compared with mammals, research on PEP of molluscs is relatively scarce at home and abroad. This observation led us to investigate PEP and whether it is related to reproductive development of mollusk. We analyzed the natural PEP of Haliotis discus hannai and the protein expression level changes during gonad development. Fluorescence quantitative PCR (qRT-PCR) and enzyme activity analysis were used to detect the expression of the PEP in tissues of H. discus hannai. The enzyme activity and the expression level of PEP were the highest in H. discus hannai gonad, followed by the muscle. PEP was purified from the gonad by column chromatography with a molecular weight of 82 ku. The isoelectric point of PEP was determined to be 5.5 by two dimensional electrophoresis, and it was similar to the isoelectric point of predicted PEP of abalone. The 182 peptides were obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, which were consistent with the H. discus hannai prolyl endopeptidase of NCBI (KY214290). The analysis of enzymatic properties showed that the optimum temperature and pH of PEP were 25 °C and 6.0, respectively, and high enzyme activity can be maintained at 15-25 °C and pH 5-8. Circular dichroism was used to analyze the effect of temperature on structure of PEP. The results showed that the secondary structure of PEP changed significantly as the temperature increased. When the temperature increases from 25 °C to 95 °C, the content of α-helix structure decreases from 12.5% to 5.8%, and the random coil increases from 24.4% to 28.0%. When the temperature was lowered from 95 °C to its optimum temperature (25 °C), the content of α-helical structure decreased by 1%, β-folding decreased by 4.6%, β-turn and random coiling structure increased by 1.5% and 2.4%, respectively, compared with the initial secondary structure at the same temperature. The secondary structure could not be restored, indicating that the thermal denaturing of PEP was irreversible. The fitting thermal denaturation temperature was (51.4±0.2) °C. In order to further analyze the protein and gene expression level of PEP in the development of H. discus hannai gonad, we divided abalone gonad development into different growth stages (preliminary, early, middle and late growth stages), and the results of Western blot and qRT-PCR analysis showed that PEP could be detected in all stages of male and female. The expression level was the highest in the middle stage of maturity of male gonad and the late stage of maturity of female gonad. These results indicated that the difference of PEP at developmental stages of H. discus hannai gonad demonstrated that it may be involved in the process of gonad development. The current study analyzed PEP of mollusks and speculated that PEP is associated with gonad development in H. discus hannai. It provides a theoretical reference for further research of the function of PEP in H. discus hannai gonad development.