Abstract: The conventional method depending on culture is known as the gold standard for microbial infection, but the long-time report cycle cannot meet the needs for early detection and early intervention. With the development of molecular biology, more and more rapid diagnostic technology based on gene and protein level are applied on the clinical microbiology laboratory. Molecular hybridization and gene chip technology can not only greatly reduce the reporting time, but also identify the differences between species. The application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry（ MALDI-TOF MS） filled the gap in detecting microbes on protein level. The next-generation sequencing technology has been widely used in the detection of pathogens in sterile body fluid. The homology analysis based on multilocus sequencing typing（ MLST） has also been applied to many studies. This review aims to introduce the rapid diagnostic technique for nosocomial infection microbes so as to provide reference for develop rapid detection technology for pathogens causing hospital-acquired nosocomial infection.