Single-molecule fluorescence technology stands at the forefront of scientific research as a sophisticated tool, pushing the boundaries of our understanding. This review comprehensively summarizes the technological advancements in single-molecule fluorescence detection, highlighting the latest achievements in the development of single-molecule fluorescent probes, imaging systems, and biosensors. It delves into the applications of these cutting-edge tools in drug discovery and neuroscience research, encompassing the design and monitoring of complex drug delivery systems, the elucidation of pharmacological mechanisms and pharmacokinetics, the intricacies of neuronal signaling and synaptic function, and the molecular underpinnings of neurodegenerative diseases. The exceptional sensitivity demonstrated in these applications underscores the vast potential of single-molecule fluorescence technology in modern biomedical research, heralding its expansion into other scientific domains.