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In this study, nanovesicles are developed using triblock polyampholytes composed of neutral poly(ethylene glycol), cationic poly(L\u2010lysine), and anionic poly(\u03b1,\u03b2\u2010aspartic acid) segments (PEG\u2010PLys\u2010PAsp) poly(aspartate) segments. By controlling the polymerization degree of these cationic and anionic segments, narrowly distributed nanovesicles are successfully assembled with a hydrodynamic diameter of \u2248140 nm. The membrane thickness of the nanovesicles is around 15\u00a0nm, corresponding to a uniform polyion complex layer. Cross\u2010linking the membrane of the nanovesicles via amide bonds enhance their stability in physiological salt and temperature conditions. In vivo, the cross\u2010linked nanovesicles exhibit prolonged blood circulation and selective accumulation in the spleen after intravenous injection in mice. 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