Two-dimensional nanomaterials (2DNMs) exhibit significant potential for the development of functional and specifically targeted biosensors, owing to their unique planar nanosheet structures and distinct physical and chemical properties. Biomodification and biomimetic synthesis offer green and mild approaches for the fabrication of multifunctional nanohybrids with enhanced catalytic, fluorescent, electronic, and optical properties, thereby expanding their utility in constructing high-performance biosensors. In this review, we present recent advances in the synthesis of 2DNM-based nanohybrids via both biomodification and biomimetic strategies for biosensor applications. We discuss covalent and non-covalent biomodification methods involving various biomolecules, including peptides, proteins, DNA/RNA, enzymes, biopolymers, and bioactive polysaccharides. The engineering of biomolecule-nanomaterial interfaces for the creation of biomodified 2DNM-based nanohybrids is also explored. Furthermore, we summarize the biomimetic synthesis of 2DNM-based bio-nanohybrids through pathways such as bio-templating, biomolecule-directed self-assembly, biomineralization, and biomimetic functional integration. The potential applications of these nanohybrids in diverse biosensing platforms-including colorimetric, surface plasmon resonance, electrochemical, fluorescence, photoelectrochemical, and integrated multimodal biosensors-are introduced and discussed. Finally, we analyze the opportunities and challenges associated with this rapidly developing field. We believe this comprehensive review will provide valuable insights into the biofunctionalization of 2DNMs and guide the rational design of advanced biosensors for diagnostic applications.