The invention provides a targeted antibacterial peptide against Gram-negative bacteria and a preparation method and an application. A sequence of the targeted antibacterial peptide is as shown in a sequence table SEQ ID No. 1, and in the preparation method, an R language is utilized for statistical analysis of amino acid sequence characteristics of a natural antimicrobial peptide library, variouspeptide chain parameters affecting antibacterial activity of a peptide chain having the antibacterial activity only against Gram-negative bacteria are statistically analyzed, and the optimal amino acid composition is obtained by screening, namely K, G, L, with the number of positive charges being 4, and the hydrophobicity being 30%-50%. Tryptophan is inserted into the center of a screening sequence to obtain a centrally symmetric short peptide sequence through design, so as to obtain a short-chain narrow-spectrum antibacterial peptide sequence which is low in toxicity and highly effective, andnot easy to cause disorder of immune regulation in a body. The targeted antibacterial peptide is beneficial to maintaining micro-ecological balance while killing harmful bacteria. The antibacterial peptide has relatively high activity, high stability, relatively low cytotoxicity, and substitute antibiotics, thereby having an application potential for becoming a safer and environmentally-friendlynovel antibacterial product.