The invention discloses protamine compounded preparation, a preparation method and application thereof. The protamine compounded preparation of the invention can be singly prepared with the protamine and chitosan, or prepared by compounding one or mixture of over two of glycine, sodium acetate, undecynol monoglyceride, acetic acid, absolute alcohol, lysostaphin, lysozyme, anti-bacterial peptide and sterilized distilled water. The method for preparing the protamine compounded preparation comprises the following steps: accurately weighting each component according to rate in aseptic or clean workshops; putting in clean glass or stainless steel, and uniformly stirring; and sealing in sterile containers. The product not only effectively kills the gram-positive cocci, such as staphyloccocus aureus rosenbach, monilia albican, neisseria gonorrhoeae, streptococcus, anaerobic bacteria, pseudomonas aeruginosa, escherichia coli, pneumococcus, D enterococcus, staphylococcus tetragenus, listeria monocytogenes, stenotrophomonas maltophilia and stomach helicobacter pylori, but also kills the gram-negative bacteria.