Antimicrobial peptides (AMPs) are expected to solve problem of antibiotic resistance because of their distinctive nonspecific membrane-disruptive mechanism. However, clinical applications of AMPs have been precluded by their poor stability, although various complex chemical strategies have been employed to solve this problem, and this undoubtedly greatly increases the manufacturing cost. Herein, a series of novel peptides with high stability were developed based on protease-specific cleavage sites and symmetrical end-tagging. Among these peptides, II-I4-II exhibited the best antibacterial activity and the highest therapeutic index. More importantly, II-I4-II showed extremely high stability in the presence of various proteases, physiological salts and serum, and under acid, alkali, and heat conditions, and it exhibited excellent therapeutic potential in vivo. Additionally, II-I4-II exhibited a membrane-disruptive mechanism and a low propensity to induce resistance. In general, these findings contribute to the design of AMPs with high stability and might accelerate clinical applications of AMPs.