In order to carry out the goal of highly sensitive, fast and accurate detection and identification of pathogen, a novel method for highly sensitive and fast detecting trace amount of nucleic acid sample is introduced. A microfluidic chip isothermal DNA amplification for bacteria molecular diagnostics is developed. New micro-nanoliter reaction assay chip is manufactured, and the surface inertia processing technique of chip is advanced to drop the chip surface adsorption to DNA molecules. A portable confocal detector with high numerical aperture and long working distance is built to reduce background fluorescence and improve the sensitivity. The sensitivity of 5 DNA copies in micro-nanoliter reaction assay is obtained. The method and detector was verified by Escherichia coli identification to acute respiratory disease, and can meet the application of low-cost detection of clinic medicine.