Abstract: The rapid identification of pathogenic microorganisms has become increasingly critical in clinical diagnostics and public health management. However, conventional detection methods are constrained by their limited analytical scope, suboptimal sensitivity, and time-consuming procedures, particularly in scenarios with unstable sample quality or low pathogen load, which frequently compromises diagnostic accuracy. In this context, high-throughput sequencing (HTS) technology has emerged as a pivotal technological approach in pathogen detection, leveraging its unparalleled throughput, ultrahigh sensitivity, and pan-pathogen detection capability. This article systematically reviews the technical principles of HTS, its specific applications in pathogen detection, and the corresponding data analysis methodologies, aiming to provide valuable insights and references for enhancing pathogen identification efficiency in clinical and epidemiological practice.