This work reported for the first time a facile template-free method to prepare polypeptide-based vesicles (peptosomes) through one-step complex self-assembly of carboxyl-terminated hyperbranched polyester and cationic poly-L-lysine (PLL). The preparation of such peptosomes, named complex peptosomes (CPs) here, is very simple just by mixing two kinds of polymer aqueous solutions together. The CP size can be readily controlled from nanosize to microsize through the adjustment of polymer concentration. The resulting nanosized CPs show unexpected size stability independent of a broad solution pH range, long-term storage stability, and almost no cytotoxicity and have demonstrated great potential to be used as the carriers in drug delivery.