Based on butterfly microfluidic gene chip technology, a method for rapid, accurate and efficient detection of 19 common pathogenic microorganisms of nosocomial infection was established, and a butterfly microfluidic gene chip with high-throughput detection was designed and fabricated. Using constant temperature amplification technology, using the polymerase with chain replacement function to react at constant temperature (65^?) and combined with microfluidic chip technology, primers were designed according to the target genes of 19 pathogenic microorganisms, and a butterfly microfluidic gene chip which can detect 19 common pathogenic microorganisms of nosocomial infection was made to simplify the inspection operation process and verify the sensitivity of the chip. The butterfly microfluidic gene chip can be used for the rapid and efficient detection of 19 common pathogenic microorganisms of nosocomial infection, and provides a new idea for the detection and auxiliary diagnosis of pathogenic microorganisms of nosocomial infection.