Since 1983, multivesicular liposomes (MVLs), as a member of the liposome family, have been of interest in the biomaterials and medical fields. MVLs have multiple aqueous compartments separated by phospholipid bilayers and an internal aqueous phase of up to 90%. They also have the advantages of reducing the number of injections, extending the duration of drug action, and improving patient compliance. So far, most of the MVLs reported in the literature are above 10 μm in size and have made good progress mainly in the encapsulation of analgesic drugs. This review provides an overview of the preparation methods, characterization methods, and drug release mechanisms of MVLs that have been reported in the literature in the last decade. There are relatively several methods for preparing MVLs, including the double emulsification method, spray atomization technique, and electroforming method. Currently, the main characterization methods used for MVLs are optical/fluorescent confocal imaging, scanning electron microscopy imaging, determination of particle size distribution, entrapment efficiency, and determination of zeta potential. Because of the large volume of the internal aqueous phase of MVLs and the high hydrophilic drug encapsulation rate of the internal vesicles, the individual vesicles gradually rupture and the hydrophilic drug gradually gets released during in vitro release, with a three-phase release pattern of sustained release. This review also summarizes the current status of clinical studies and types of commercialized products. At present, the application of MVLs regarding analgesics has reached stages II-IV, and three commercialized formulations have entered the clinic with satisfactory results. Moreover, this review summarizes the current progress in applied research, mainly in the delivery of anticancer drugs, analgesic drugs, and protein peptides. Last but not least, the challenge and prospects regarding small-sized MVLs, diverse biomedical applications, and scale-up strategies are proposed.