Abstract: To explore the preparation, enrichment, and structural analysis of bitter almond antimicrobial peptides, in this experiment, the protein was extracted from the defatted bitter almonds, and the protein was hydrolyzed by enzymes to prepare bitter almond protein antibacterial peptides. The preparation process was optimized, and the resulting peptides' antibacterial activity was detected. The peptides were further purified and enriched. The components with the strongest antibacterial activity were isolated, and their structure was analyzed. The results showed that the papain hydrolysate had the strongest bacteriostatic effect on Gram-positive bacteria, especially Staphylococcus aureus. The optimal conditions for the preparation of bitter almond antibacterial peptides were as follows: The enzymatic hydrolysis temperature was 74℃, the enzyme-to-substrate ratio was 2.5%, and the pH was 7. The protein concentration of the antibacterial peptides was 19.21%, and the minimum inhibitory concentration was 3.13 mg/mL. The antibacterial peptides were isolated and purified by ultrafiltration and gel filtration chromatography (Sephadex G-25). Peptide component A-II-b was screened out to have the strongest inhibitory activity against Staphylococcus aureus. The gel filtration conditions were optimized with a sample concentration of 9.38 mg/mL, an elution rate of 0.68 mL/min, and pure water as the eluent. Under these conditions, the peak area ratio of peptide component A-II-b was 13.15%. The structure of peptide component A-II-b was analyzed by liquid chromatography-mass spectrometry, and the results were imported into the UniProt database and the APD3 database for matching. Seven kinds of bitter almond peptides with potential antibacterial activity with molecular weights of 1500~2500 Da were screened out, including ALPDEVLQNAFRIS, ESWNPRDPQFQWAGVA, VAYWSYNNGEQPLVA, FLDLSNDQNQLDQVPR, GENDNRNQIIRVR, RNLQGENDNRNQIIRVR, and RALPDEVLQNAFRIS. This study promoted the in-depth development and resource reuse of almond active peptides and also provided a theoretical basis for their research and further development and utilization as an antibacterial peptide.