Membrane lytic peptides (MLP) are widely explored as cellular delivery vehicles or antitumor/antibacterial agents. However, the poor selectivity between cancer and normal cells slims their prospects as potential anti‐tumor drugs. Herein, we have developed a rationally designed self‐assembly strategy to enhance tumor selectivity of MLP‐based conjugates, incorporating a hydrophobic triphenylphosphonium (TPP) group for mitochondria targeting, and a hydrophilic arginine‐glycine‐aspartic acid (RGD) sequence targeting integrins. The self‐assembly nanoparticles can enhance the stability of the peptides in vitro plasma and be endocytosed selectively into the cancer cells. The histidine‐rich lytic peptide component assists the disruption of endosomal/lysosomal membranes and subsequent the mitochondria membrane, which leads to apoptosis. This rational design of MLP‐based conjugates provides a practical strategy to increase the application prospects of lytic peptides in cancer treatment.