Biomimetic giant membrane vesicles, with size and lipid compositions comparable to cells, have been recognized as an attractive experimental alternative to living systems. Due to the similarity of their membrane structure to that of body cells, cell-derived giant plasma membrane vesicles have been used as a membrane model for studying lipid/protein behavior of plasma membranes. However, further application of biomimetic giant membrane vesicles has been hampered by the side-effects of chemical vesiculants and the utilization of osmotic buffer. We herein develop a facile strategy to derive giant membrane vesicles (GMVs) from mammalian cells in biofriendly medium with high yields. These GMVs preserve membrane properties and adaptability for surface modification and encapsulation of exogenous molecules, which would facilitate their potential biological applications. Moreover, by loading GMVs with therapeutic drugs, GMVs could be employed for drug transport to tumor cells, which represents another step forward in the biomedical application of giant membrane vesicles. This study highlights biocompatible GMVs with biomimicking membrane surface properties and adaptability as an ideal platform for drug delivery strategies with potential clinical applications.