Abstract: Objective Mutations in mitochondrial 12 S rRNA gene are the molecular basis of hearing loss, of these, the A1555 G and C1494 T mutations have been found to be associated with nonsyndromic hearing loss in many families worldwide.This study aimed to establish a novel method for detection the deafness-associated mitochondrial 12 S rRNA A1555 G or C1494 T mutation. Methods We used 3 standard templates (wild type, A1555 G, C1494T mutation) of human mitochondrial 12 S rRNA to design four primers for PCR amplification by employing the Primer 5.0 software, then this method was applied to clinical screening of 200 non-syndromic hearing loss subjects and confirmed by Sanger sequencing. Results We found that samples with the A1555 G mutation can be detected by two specific bands (226 bp and 736 bp), samples with the C1494 T mutation can be detected by the 488 bp and 736 bp, while the samples without these mutations can be detected by only one band (736 bp).Using this method, we detected the 8 cases carrying these mutations (4%, 8/200), including 4 cases with A1555 G mutation, 4 cases with C1494 T mutation, which was well consistent with the results of DNA sequencing (Kappa=1.000, P < 0.01). Conclusion This is a convenience, cost-effective, accurate and useful method for detection the deafness-associated mitochondrial A1555 G or C1494 T mutation, which could identify the subjects at risk and effectively prevent the aminoglycoside induced hearing loss.