Biosensors featuring single molecule detection present huge opportunities as well as challenges in food safety inspection, disease diagnosis, and environmental monitoring. Single-molecule detection is largely lacking of high enough activity, precision molecule selectivity, and understanding in the exact operating mechanism. Single-atom catalysts (SACs), especially those metals-nitrogen-carbon that mimic the natural metalloenzyme structure, and with well-defined metal atom bond configurations, high level of molecular selectivity, and easy fabrication, endow single molecule detections with practical-use feasibilities. The recent advances in singleatom catalysts also present new pathways in the key mechanism understandings. In this short review, we will first visit the brief history and advantages of SACs that have been explored only recently for moleculescale biosensors, where they are analogous and also differentiated from those nanozymes and natural metalloenzymes. Their applications in electrochemical, photochemical, and photoelectrochemical sensors are then discussed comprehensively by focusing on the different molecule-scale sensing modes in achieving local coordination-modulated signal amplifications. Finally, we identify new opportunities and challenges faced by these SACs-based single molecule detections in the further development of biosensors.