The present invention relates to cell membrane-derived nanovesicles, a method of preparing the same, and a pharmaceutical composition and a diagnostic kit using the nanovesicles. The cell membrane-derived nanovesicles according to the present invention may prevent the occurrence of potential side effects because intracellular materials (e.g., genetic materials and cytosolic proteins) unnecessary for delivery of therapeutic or diagnostic substances are removed from the nanovesicles. In addition, since the nanovesicles may be targeted to the specific types of cells or tissues, therapeutic or diagnostic substances may be predominantly delivered to the targeted cells or tissues while delivery of therapeutic or diagnostic substances to untargeted sites may be inhibited. Therefore, when the cell membrane-derived nanovesicles are applied to disease treatment, the side effects of therapeutic substances such as drugs may be reduced, so that suffering and inconvenience of patients may be alleviated during the course of treating diseases, and therapeutic efficacy may be improved. In addition, the cell membrane-derived nanovesicles of the present invention, in which substances for the treatment or diagnosis of diseases are loaded, and a method of preparing the nanovesicles may be used in vitro or in vivo for therapeutic or diagnostic purposes, or for experimental use.