In the family of Janus nanomaterials, Janus nanosheets possess not only the advantages of Janus nanomaterials, but also the advantages of two-dimensional nanosheets, endowing them with many extraordinary properties. Therefore, Janus nanosheets have great potential in the fields of interfacial engineering, catalysis, and molecular recognition. This review summarizes and discusses the recent advances in both the preparation and applications of freestanding Janus nanosheets. After a short introduction to different types of Janus nanosheets, a variety of methods for preparing freestanding Janus nanosheets are introduced, including the surface reaction, interface reaction, emulsion reaction, self-assembly, and stripping of non-Janus nanosheets, as well as selective grafting of existing Janus nanosheets. Then, the wide applications of Janus nanosheets in the fields of emulsification, catalysis, polymer reinforcement, nanomotors, and molecular recognition are summarized in detail. Finally, a discussion on the remaining challenges and future perspectives in this field is included. This review will not only deepen the understanding of Janus nanosheets, but also benefit the designs and fabrications of extraordinary and multi-functional Janus nanosheets.