Chitosan (CS) and two-dimensional nanomaterial (2D nanomaterials)-based scaffolds have received widespread attention in recent times in biomedical applications due to their excellent synergistic potential. CS has garnered much attention as a biomedical scaffold material either alone or in combination with some other material due to its favorable physiochemical properties. The emerging 2D nanomaterials, such as black phosphorus (BP), molybdenum disulfide (MoS), etc., have taken huge steps towards varying biomedical applications. However, the implementation of a CS-2D nanomaterial-based scaffold for clinical applications remains challenging for different reasons such as toxicity, stability, etc. Here, we reviewed different types of CS scaffold materials and discussed their advantages in biomedical applications. In addition, a different CS nanostructure, instead of a scaffold, has been described. After that, the importance of 2D nanomaterials has been elaborated on in terms of physiochemical properties. In the next section, the biomedical applications of CS with different 2D nanomaterial scaffolds have been highlighted. Finally, we highlighted the existing challenges and future perspectives of using CS-2D nanomaterial scaffolds for biomedical applications. We hope that this review will encourage a more synergistic biomedical application of the CS-2D nanomaterial scaffolds and their utilization clinical applications.