The invention relates to an ultrafiltration membrane separation and peptide sequence identification method of bovine marrow polypeptides based on response surface optimization. According to the method, firstly, protein extraction process parameters are reasonably screened and determined, secondly, a protein extract is subjected to ultrafiltration membrane separation through a response surface optimization method. By adopting the method, the protein content is 76.9mg/mL, the separated part is further purified with ionic exchange resin, the polypeptides are subjected to structure characterization and peptide sequence identification with infrared spectrum, a scanning electron microscope, a two-dimensional electrophoresis technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the like, totally 20 kinds of active peptide sequences are identified, including 10 kinds of antioxidative peptides, 7 kinds of antibacterial peptides and 3 kinds of antihypertensive peptides. The method has the advantages of short extraction time, simple operation, reliable identification technology, high efficiency and the like, is easy for industrial production, and provides basic reference to development and comprehensive utilization of animal marrow-derived antibacterial, antioxidative, antihypertensive and other active polypeptides.