The long-dreamed-of capability of monitoring the molecular machinery in living systems has not been realized yet, mainly due to the technical limitations of current sensing technologies. However, recently emerging quantum sensors are showing great promise for molecular detection and imaging. One of such sensing qubits is the nitrogen−vacancy (NV) center, a photoluminescent impurity in a diamond lattice with unique roomtemperature optical and spin properties. This atomic-sized quantum emitter has the ability to quantitatively measure nanoscale electromagnetic fields via optical means at ambient conditions. Moreover, the unlimited photostability of NV centers, combined with the excellent diamond biocompatibility and the possibility of diamond nanoparticles internalization into the living cells, makes NV-based sensors one of the most promising and versatile platforms for various life-science applications. In this review, we will summarize the latest developments of NV-based quantum sensing with a focus on biomedical applications, including measurements of magnetic biomaterials, intracellular temperature, localized physiological species, action potentials, and electronic and nuclear spins. We will also outline the main unresolved challenges and provide future perspectives of many promising aspects of NV-based bio-sensing.