Two‐dimensional (2D) metal‐organic framework (MOF) nanosheets, as an emerging type of 2D materials, attract numerous attention due to their unique properties. First, the ultrathin thickness and nanoscale of the materials results in homogeneous dispersion in aqueous solution, giving the materials more opportunities to be utilized in solution chemistry, especially beneficial to the biomimetic catalysis and bio‐related analytical applications. Second, the large surface area and accessible active sites of the MOF nanosheets are favorable to the binding between materials and the substrate, leading to their superior performance in catalysis, sensing and enzyme inhibition. Third, the suitable sizes of nanopores on the 2D MOF nanosheets give them the abilities to act as membranes for highly selective and energy‐saving gas separation. This minireview covers the synthesis, characterization as well as bio‐related and separation applications of 2D MOF nanosheets. Nanosheets: Two‐dimensional (2D) metal‐organic frameworks (MOFs) nanosheets, as an emerging type of 2D materials, are attracting increasing applications in catalysis, sensing, enzyme inhibition, MALDI‐TOF matrix and gas separation. This Minireview summarizes 2D MOF nanosheets and their diverse applications with the integration of the fundamental structures and their application mechanism.