Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity, specificity, and low consume of sample and reagent is keyword to low cost molecular diagnostics. In this paper, a sensitive DNA isothermal amplifi-cation method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate (PC) microfluidic chip. A portable confocal optical fluo-rescence detector was specifically developed for the microfluidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background. The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65 aM, and a detection limit of less than five copies of genomic DNA. Contrast to the general polymerase chain reaction (PCR) at eppendorf (EP) tube, the detection time in our developed method was reduced from 1.5 h to 45 min for multi-target parallel detection, the consume of sample and reagent was dropped from 25 L to 1.45 L. This novel microfluidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets, with potential applications in biotechnology, medicine, and clinical molecular diagnostics.