Abstract: Next-generation sequencing technology is a revolutionary genomic sequencing method capable of simultaneously sequencing millions to billions of DNA or RNA molecules. In recent years, due to its high-throughput, rapid, and highly sensitive characteristics, this technology has been widely applied in the clinical diagnosis of various infectious diseases. Compared with traditional pathogen culture methods, Next-generation sequencing can rapidly and comprehensively detect multiple, unknown, or rare pathogens, provide complete genomic information, and monitor the transmission dynamics and mutation trends of pathogens in real time. It can also identify mutation sites associated with drug resistance, offer more comprehensive drug resistance information, conduct holistic analysis of complex microbial communities, trace the transmission routes of pathogens. Despite its significant advantages, Next-generation sequencing currently faces a series of issues such as high costs, short sequencing reads, a lack of standardized procedures, and privacy breaches. In the future, the development of Next-generation sequencing should focus on further shortening sequencing time, reducing sequencing costs, extending sequencing reads, and improving standardized procedures. Additionally, it can be combined with artificial intelligence for comprehensive and automated analysis.