Antimicrobial resistance poses a serious threat to human health and is the main cause for the increase in infection-related mortality. Rational and timely use of antimicrobial agents is crucial for improving the prognosis of infected patients, however, overdose and inappropriate use of antimicrobial agents can accelerate the acquisition of drug resistance of pathogenic microorganisms, promote the emergence of new resistance mechanisms, and ultimately lead to the emergence of "superbugs". Therefore, early identification of antimicrobial resistance is of significant importance for the rapid diagnosis and precise treatment of infectious diseases. With the development of molecular dia-gnostic techniques, third-generation sequencing (TGS) technique offers innovative solutions for detecting microbial resistance due to its advantages in long read length, easy operation, and rapid detection, significantly promoting early diagnosis and real-time monitoring of antimicrobial resistance. This review systematically summarizes the research progress of TGS in microbial resistance detection, analyzes its advantages and limitations in rapid detection of resis-tance in viruses, bacteria, and fungi, thus provides new perspectives for the early diagnosis and treatment of pathogenic microbial infections.