Abstract: Objective：To establish a rapid method for the detection of mitochondrial 12S rRNA gene 1494C>T and 1555A>G mutations related to drug-induced deafness based on high resolution melting (HRM) analysis. Methods：Mutational plasmid DNA standard materials were constructed by the site directed mutagenesis cloning strategy. The PCR-HRM system was developed to detect the mitochondrial 12S rRNA gene mutations of 106 non-syndromic deafness patients, and the results were further verified with DNA sequencing. Results：The established method could detect the mitochondrial 12S rRNA gene 1494C>T and 1555A>G mutations correctly, and the HRM curves of each genotype were obvious and easy to be analyzed. Among 106 patients with non-syndromic deafness, 6 were detected A1555G mutation, and the results were consistent with those of DNA sequencing. Conclusion：The HRM method for the detection of mitochondrial 12S rRNA gene 1494C>T and 1555A>G mutations related to drug-induced deafness is successfully established, which is simple, rapid and accurate, and may be applied to the mutation screening and clinical molecular diagnosis.