Abstract: Background In recent years, natural bioactive peptides have garnered significant attention due to their high safety profile and diverse biological activities, demonstrating immense potential particularly in the field of immunomodulation. The search for safe and effective novel natural immune regulators has become a research hotspot. Objective This paper systematically reviews recent advances in preparation methods and separation/purification technologies for immunomodulatory peptides, focusing on the latest research findings from plant, terrestrial animal, and marine sources to provide references for developing novel, safe, and highly effective natural immunomodulators. Progress Preparation methods for immunomodulatory peptides primarily include enzymatic hydrolysis (via targeted protease digestion), microbial fermentation (utilizing enzyme-producing microorganisms), and chemical synthesis (enabling precise sequence control). For separation and identification, membrane separation technology and various chromatographic techniques are widely applied for peptide enrichment and purification. Currently, peptides with immunomodulatory activity have been screened from diverse protein sources, with marine peptides garnering significant attention due to their novel structures and diverse activities. Mechanistic studies indicate these peptides exert immunoregulatory effects by modulating macrophage function, regulating cytokine secretion, and activating signaling pathways such as NF-κB and MAPK through TLR2 and TLR4/MD-2 receptors. Conclusion Immunomodulatory bioactive peptides represent a class of natural functional molecules with significant development potential, offering broad application prospects in functional foods, pharmaceuticals, and aquaculture. Future efforts should focus on enhancing structure-activity relationship studies, elucidating action mechanisms, and developing delivery systems to advance their practical application in functional foods and pharmaceuticals.