Single molecule biological detection technology is an efficient technology to understand the dynamic characteristics of various biomolecules at the single molecule level and explore their structure and function. The advantage of this technology is that it can detect the heterogeneity of free energy on a single molecule, which is beyond the traditional methods. Therefore, researchers use it to solve long-standing problems in complex biological systems, heterogeneous catalysis, biomolecular interactions, enzyme systems and conformational changes. In terms of medical detection, detecting specific information about single molecules or their interactions with biological factors is not only crucial for the early diagnosis and treatment of various diseases such as cancer, but also has great potential for real-time detection and precision medicine. The advantages of high specificity and high precision of single-molecule bioassays are used to real-time detection of single biomolecules in molecular populations, and can be combined with multiple high-throughput analysis for the precise diagnosis of clinical samples. In this paper, the principle of single molecule detection and the application of biosensing are introduced, and the detection methods and related applications are summarized. Finally, the prospect and development direction of this research direction are discussed.