By studying the principles of self-assembly and combining the structural parameters required for the asymmetric distribution of antimicrobial peptides (AMPs), we newly designed and screened the high-activity and low-toxicity AMP F2I-LL. This peptide can form a supramolecular hydrogel with a nanofiber microstructure in a simulated physiological environment (phosphate buffered saline), which exhibits broad-spectrum antibacterial activity. Compared with monomeric peptides, the introduction of a self-assembly strategy not only improved the bactericidal titer but also enhanced the serum stability of AMPs. Mechanistic studies showed that the positive charge enriched on the surface of the nanofiber was conducive to its rapid binding to the negatively charged part of the outer membrane of bacteria and further entered the inner membrane, increasing its permeability and ultimately leading to cell membrane rupture and death. This work provides insights into the design of nanopeptides with broad-spectrum antibacterial activity and provides new results for the development of biomedicine.