Sepsis is a vital disorder that threatens human life and health. Early and rational antibiotic use is essential for sepsis treatment. However, due to the wide variety of pathogens, rapid, comprehensive, and accurate identification of pathogens is a difficult task in diagnosis and treatment of sepsis. Traditional pathogenic surveillance often leads to delayed treatment or inappropriate antimicrobial therapy due to a narrow detection range, single target, low accuracy, low positive rate, and long detection period, making it challenging to meet the clinical needs of identifying the pathogens of serious infections. With its advantages of high throughput, comprehensive coverage, and high accuracy, mNGS can directly analyze the spectrum, abundance, and distribution of pathogenic microorganisms in specimens without bias and has shown great promise in the diagnosis and treatment of severe and complicated infectious diseases, such as identification of unknown pathogens, and drug resistance gene monitoring. This paper summarizes and reviews diagnostic applications of mNGS in various infectious diseases, the factors affecting detection and rational application in the pathogen diagnosis in sepsis.