Antimicrobial peptides can not only be used as biological preservatives to prevent microbial corruption in food processing and storage, but also be expected to alleviate the problem of drug resistance caused by antibiotic abuse because of its high antibacterial activity and unique antibacterial mechanism. To obtain a novel antimicrobial peptide, the sequence length, charge numbers and amino acid composition of this antimicrobial peptides were determined and optimized based on the existing database of antimicrobial peptides. According to the bioinformatics prediction, analysis tools and rational de novo design, the novel antimicrobial peptide YHX-1 was designed with the sequence of GKLLSKLLKKLLK. The antibacterial activity of YHX-1 against gram-positive and gram-negative bacteria was evaluated by agar diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration and bactericidal kinetic curves. Its stability in salt ion, plasma and slightly acidic environment, secondary structure, hemolytic activity and cytotoxicity were also determined. The results showed that YHX-1 showed good antibacterial activity against six kinds of pathogenic bacteria with 4μg/mL of MIC against Listeria monocytogenes and Salmonella typhimurium, and it could cause a mortality rate of more than 99.99% of Listeria monocytogenes within 2h. YHX-1 showed a certain sensitivity for salt ions (200mmol/L Na⁺ or 2mmol/L Ca²⁺) and 10%-25% plasma which the MIC increased by 2 to 8 times. Furthermore, the MIC of YHX-1 was not significantly affected by slightly acidic environment (pH 5.4-7.4). The results of circular dichroism spectrum showed a typical α-helix structure of YHX-1. Hemolytic activity and cytotoxicity test showed that YHX-1 had good cell selectivity. The newly designed antibacterial peptide YHX-1 was efficient broad-spectrum bactericidal, highly bactericidal efficient and safe which could be used as a new antibacterial agent for food preservation. The de novo design strategy could also provide a reference for designing antimicrobial peptides.