Objective:To investigate the mutation of non-syndromic sensorineural hearing loss (NSHL) and identify its molecular etiopathogenisis. Methods:Peripheral blood samples were obtained from 22 NSHL patients collected by out-patient clinic. Their genomic DNA were extracted from peripheral blood by extraction kits to undergo polymerase chain reaction and sequencing so as to detect the mutations of GJB2,SLC26A4 and mitochondrial 12SrRNA gene. Results:Of 22 patients,three mutations of GJB2 gene were found:2 have GJB2 235delC heterozygous mutation,2 have 235delC homozygous mutation and 1 have 176del16bp+299delAT double mutation;2 mutaions was found out in MtDNA 12SrRNA gene:C1494T and A1555G;SLC26A4 gene have 1 IVS7-2 homozygous and 2 heterozygous mutations. Conclusion:The PCR combined DNA sequencing is a classic and effective method in genetic diagnostic of hereditary deafness,able to identify the molecular etiopathogenisis of NSHL. But this method exist many shortcomings,such as unable to qualitate,time and money consuming. Furthermore,it can not detect multiple mutations of different genes at the same time. Therefore,a new method,which has advantages in genetic diagnosis of NSHL,such as low time and money consuming,high performance and accuracy,must to be developed. These advantages make it fit to be used in clinic gene testing of hereditary deafness.