Nanomaterials-based artificial enzymes (nanozymes) with valuable enzyme-like catalytic properties have been booming during the past few years. Promoted by the advances in biological medicine and nanotechnology, nanozymes possess the potential to serve as an emerging agent for biosensing, immunoassays, detection and diagnosis, catalytic therapeutics, and other applications in the biomedicine field. Two-dimensional (2D) nanomaterials are of considerable interest in biomedical applications due to their ultrathin layered structure and unique physiochemical properties. Inspired by the diversified catalytic performance of 2D nanomaterials, scientists extensively have developed 2D materials as bioactive nanozymes for theranostic nanomedicine. Here, recent advances in enzyme-like 2D nanomaterials design and construction are comprehensively presented. Additionally, we exhibit that, with the synergistic effect of catalytic activities and desirable physicochemical performances, 2D nanozymes can serve as versatile platforms with extensive applications from target detection to in vivo theranostic. It is believed that such promising alternatives towards natural enzymes will be of vital significance in the field of nanotechnology and biomedicine.