Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 10(2) CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA.