Abstract: The abalone (Haliotis discus hannai) mantle processing by-products was used to prepare biologically active peptides with inhibiting the activity of angiotensin converting enzyme (ACE), which provided a new idea for the high-value utilization of abalone processing by-products. The serine protease prepared from the digestive glands of Litopenaeus vannamei was used to enzymatically hydrolyze the mantle proteins of Haliotis discus hannai. The ACE inhibitory activity of the enzymatic hydrolysate was used as the evaluation index to optimize the digestion conditions. After the enzymatic hydrolysis solution passed through the 3 kDa ultrafiltration membrane, the active components were separated and purified by Superdex peptide 10/300 GL gel filtration column and reversed-phase high performance liquid chromatography (RP-HPLC). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify the amino acid sequence of the purified peptide. The results showed that the ACE inhibition rate of the enzymatic hydrolysate was 13.42%. After preliminary separation by the 3 kDa ultrafiltration membrane, the ACE inhibition rate of the fractions less than 3 kDa at the same concentration was 53.25%. After further purification by gel filtration column and RP-HPLC, five peptides were identified from the peak with the highest ACE inhibitory activity. The sequences were LGDSFYYGK, LVNEVTEFAK, VDEVGGEALGR, MFLSFPTTK, VATVSLPR, indicating that the active peak was a mixture of peptides. Preparation of ACE inhibitory peptides using abalone mantle could provide a theoretical reference for the high-value utilization of the processing by-products of abalone and prawn.