As one kind of redox active layered transition-metal dioxide nanomaterials, single-layer manganese dioxide (MnO2) nanosheets have gained significant research attention in the fields of biosensing and biomedicine because of their large surface area, intense and broad optical absorption, strong oxidation ability, catalytic activity, and robust mechanical properties. This review provides a brief overview of the recent advances in the development of MnO2 nanosheet-based biosensors, bioimaging as well as drug delivery for cancer therapy. The methodologies for the preparation of MnO2 nanosheets are summarized, followed by an introduction of the nanostructure and properties of MnO2 nanosheets. Special attention is paid to their applications in biosensing, bioimaging and cancer therapy. Future perspectives and the challenges of high-performance MnO2 nanosheets are also discussed.