Mitochondrial disorders are a group of complex and heterogeneous diseases that may be caused by molecular defects in mitochondrial genomes. Pathogenic mitochondrial DNA (mtDNA) mutations are usually present in the heteroplasmic form. The degree of mtDNA mutation heteroplasmy varies among different tissues. Thus, it is important to detect and quantify the degree of mutation heteroplasmy of mtDNA. Currently, more than 100 pathogenic mtDNA point-mutations have been documented and new ones are still being extensively explored. Among them, A3243G mutation, which causes the myopathy encephalopathy lactic acidosis and stroke (MELAS) syndrome and diabetes, attracts considerable interest recently. In this study, a new microfluidic system capable of point-mutation detection of mtDNA has been demonstrated. The entire process including cell lysis, enzyme digestion, amplification of target fragment of mtDNA and optical detection can be automatically performed. Experimental results showed that the point-mutation of mtDNA can be successfully detected.