Covalent organic frameworks (COFs) are an emerging class of organic crystalline polymers with welldefined molecular geometry and tunable porosity. COFs are formed via reversible condensation of lightweight molecular building blocks which dictate its geometry in two or three dimensions. Among COFs, two-dimensional COFs (2D COFs) have garnered special attention due to their unique structure composed of two-dimensionally extended organic sheets stacked in layers generating periodic columnar π-arrays, functional pore space and their ease of synthesis. These unique features in combination with their low density, high crystallinity, large surface area, and biodegradability have made them an excellent candidate for a plethora of applications ranging from energy to biomedical sciences. In this article, the evolution of 2D COFs is briefly discussed in terms of different types of chemical linkages, synthetic strategies of bulk and nanoscale 2D COFs, and their tunability from a biomedical perspective. Next, the recent advances of these 2D nanomaterials in biomedicine and biotechnology are summarized emphasizing the principles and strategies involved. In addition, current challenges and emerging approaches of 2D COFs for the advanced biomedical applications are discussed.