The unique nano–bio interfacial phenomena play a crucial role in the biosafety and bioapplications of nanomaterials. As a representative two-dimensional (2D) nanomaterial, molybdenum disulfide (MoS2) has shown great potential in biological applications due to its low toxicity and fascinating physicochemical properties. This review aims to highlight the nano–bio interface of MoS2 nanomaterials with the major biomolecules and the implications of their biosafety and novel bioapplications. First, the nano–bio interactions of MoS2 with amino acids, peptides, proteins, lipid membranes, and nucleic acids, as well as the associated applications in protein detection, DNA sequencing, antimicrobial activities, and wound-healing are introduced. Furthermore, to facilitate broader biomedical applications, we extensively evaluated the toxicity of MoS2 and discussed the strategies for functionalization through interactions among MoS2 and the variety of macromolecules to enhance the biocompatibility. Overall, understanding the nano–bio interface interaction of two-dimensional nanomaterials is significant for understanding their biocompatibility and biosafety, and further provide guidance for better biological applications in the future.